Methods and Compositions for F-18 Labeling of Proteins, Peptides and Other Molecules

ABSTRACT

The present application discloses compositions and methods of synthesis and use of F-18 labeled molecules of use, for example, in PET imaging techniques. In particular embodiments, the labeled molecules may be peptides or proteins, although other types of molecules including but not limited to aptamers, oligonucleotides and nucleic acids may be labeled and utilized for such imaging studies. In preferred embodiments, the F-18 label may be conjugated to a targeting molecule by formation of a metal complex and binding of the F-18-metal complex to a chelating moiety, such as DOTA, NOTA, DTPA, TETA or NETA. In other embodiments, the metal may first be conjugated to the chelating group and subsequently the F-18 bound to the metal. In other preferred embodiments, the F-18 labeled moiety may comprise a targetable conjugate that may be used in combination with a bispecific or multispecific antibody to target the F-18 to an antigen expressed on a cell or tissue associated with a disease, medical condition, or pathogen. Exemplary results show that F-18 labeled targetable conjugate peptides are stable in human serum at 37° C. for several hours, sufficient time to perform PET imaging analysis.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/112,289, filed Apr. 30, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 11/960,262, filed Dec. 19, 2007, whichclaimed the benefit under 35 U.S.C. § 19(e) of Provisional U.S. PatentApplication No. 60/884,521, filed Jan. 11, 2007, each of which isincorporated herein by reference in its entirety.

FIELD

In certain embodiments, the present invention concerns a simple methodof labeling peptides with F-18, which are of use for in-vivo imaging.The preferred specific activity of the F-18 labeled peptide would beabout 1,000 to 2,000 Ci/mmol at the time of administration to thepatient. Specific activities that are in the range of 100 to tens ofthousands of Ci/mmol would also be of use. Although higher specificactivities are preferred for certain imaging applications, in otheralternative embodiments a lower specific activity of a metal-F-18complex with NOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid) oranother chelating moiety could be of use, for example, as a renal flowimaging agent or for heart and brain imaging agents to image blood flow.Preferably, F-18 labeling is accomplished without need for apurification step to separate unlabeled from labeled peptide. Morepreferably, F-18 labeled peptides are stable under in vivo conditions,such as in human serum.

BACKGROUND

Positron Emission Tomography (PET) imaging provides high resolution andquantitation from the PET images. Peptides or other small molecules canbe labeled with the positron emitters ¹⁸F, ⁶⁴Cu, ⁶⁶Ga, ⁶⁸Ga, ⁷⁶Br,^(94m)Tc, ⁸⁶Y, and ¹²⁴I to name a few. The positron emitted from thenucleus of the isotope is ejected with different energies depending onthe isotope used. When the positron reacts with an electron two 511 keVgamma rays are emitted in opposite directions. The energy of the ejectedpositron controls the average distance that a positron travels before itis annihilated by hitting an electron. The higher the ejection energythe further the positron travels before the collision with an electron.A low ejection energy for a PET isotope is desirable to minimize thedistance that the positron travels from the target site before itgenerates the two 511 keV gamma rays that are imaged by the PET camera.Many isotopes that emit positrons also have other emissions such asgamma rays, alpha particles or beta particles in their decay chain. Itis desirable to have a PET isotope that is a pure positron emitter sothat any dosimetry problems will be minimized.

The half-life of the isotope is also important, since the half-life mustbe long enough to attach the isotope to a targeting molecule, analyzethe product, inject it into the patient, and allow the product tolocalize, clear from non-target tissues and then image. If the half-lifeis too long the specific activity may not be high enough to obtainenough photons for a clear image and if it is too short the time neededfor manufacturing, commercial distribution and biodistribution may notbe sufficient. F-18 (β⁺635 keV 97%, t_(1/2) 110 min) is one of the mostwidely used PET emitting isotopes because of its low positron emissionenergy, lack of side emissions and suitable half-life. The F-18 isproduced with a high specific activity. When an isotope is attached to amolecule for targeting it is usually accompanied by some unreactedtargeting agent, which is often present in a large molar excess comparedto the radiolabeled product. Usually, the labeled product and theunlabeled product can compete for the same target in-vivo so thepresence of the cold targeting agent lowers the effective specificactivity of the targeting agent. If the F-18 is attached to a moleculewhich has a very high uptake such as 2-fluoro-2-deoxy glucose (FDG) theneffective specific activity is not as important. However, if one istargeting a receptor with a labeled peptide or performing an immunoPETpretargeting study with a limited number of binding sites available, thecold targeting agent could potentially block the uptake of theradiolabeled targeting agent if the cold targeting agent is present inexcess.

Conventional F-18 labeling of peptides involves the labeling of areagent at low specific activity, HPLC purification of the reagent andthen conjugation to the peptide of interest. The conjugate is oftenrepurified after conjugation to obtain the desired specific activity oflabeled peptide. An example is the labeling method of Poethko et al. (J.Nucl. Med. 2004; 45: 892-902) in which 4-[¹⁸F]fluorobenzaldehyde isfirst synthesized and purified (Wilson et al, J. Labeled Compounds andRadiopharm. 1990; XXVIII: 1189-1199) and then conjugated to the peptide.The peptide conjugate is then purified by HPLC to remove excess peptidethat was used to drive the conjugation to completion. The two reactionsand purification would not be a problem if F-18 had a long half-life.However the half-life of F-18 is only 2 hr so all of the manipulationsthat are needed to attach the F-18 to the peptide are a significantburden.

These methods are tedious to perform and require the use of equipmentdesigned specifically to produce the labeled product and/or the effortsof specialized professional chemists. They are not kit formulations thatcould routinely be used in a clinical setting. A need exists for arapid, simple method of 18-F-labeling of targeting moieties, such asproteins or peptides, that results in targeting constructs of suitablespecific activity and in vivo stability for detection and/or imaging,while minimizing the requirements for specialized equipment or highlytrained personnel and reducing operator exposure to high levels ofradiation. A further need exists for prepackaged kits that could providecompositions required for performing such novel methods.

SUMMARY

Fluoride binds to practically all other elements and some of those bondsare relatively stable. Peptides, bearing metal binding ligands, areknown to bind radiometals stably and at very high specific activity. Theapproach utilized in the present method was to first bind the F-18 to ametal and then chelate the F-18 metal complex with a ligand on thepeptide. The question was then, which metal (or other element e.g.boron) to choose. The elements in group IIIA (boron, aluminum, gallium,indium, and thallium) were the first choice based on a quick search ofthe literature. Lutetium may also be of use.

Alternatively, one might attach the metal or other atom to the peptidefirst and then add the F-18. The second approach might work better, forexample, for a boron fluoride connection.

Aluminum fluoride complexes are reported to be stable in-vitro (Martinezet al, Inorg. Chem. 1999; 38: 4765-4660; Antonny et al. J. Biol. Chem.1992; 267: 6710-6718). Aluminum fluoride becomes incorporated into boneand into the enamel of teeth so the complexes can also be stable in-vivo(Li, Crit. Rev. Oral Biol. Med. 2003; 14: 100-114).

The skilled artisan will realize that virtually any delivery moleculecan be used to attach the F-18 for imaging purposes, so long as itcontains derivatizable groups that may be modified without affecting theligand-receptor binding interaction between the delivery molecule andthe cellular or tissue target receptor. Although the Examples belowconcern F-18 labeled peptide moieties, many other types of deliverymolecules, such as oligonucleotides, hormones, growth factors,cytokines, chemokines, angiogenic factors, anti-angiogenic factors,immunomodulators, proteins, nucleic acids, antibodies, antibodyfragments, drugs, interleukins, interferons, oligosaccharides,polysaccharides, lipids, etc. may be F-18 labeled and utilized forimaging purposes. Similarly, the type of diseases or conditions that maybe imaged is limited only by the availability of a suitable deliverymolecule for targeting a cell or tissue associated with the disease orcondition. Many such delivery molecules are known, as exemplified in theExamples below. For example, any protein or peptide that binds to adiseased tissue or target, such as cancer, may be labeled with F-18 bythe disclosed methods and used for detection and/or imaging. In certainembodiments, such proteins or peptides may include, but are not limitedto, antibodies or antibody fragments that bind to tumor-associatedantigens (TAAs). Any known TAA-binding antibody or fragment may belabeled with F-18 by the described methods and used for imaging and/ordetection of tumors, for example by PET scanning or other knowntechniques.

In certain Examples below, the exemplary F-18 labeled peptides may be ofuse for imaging purposes as targetable constructs in a pre-targetingmethod, utilizing bispecific or multispecific antibodies or antibodyfragments. In this case, the antibody or fragment will comprise one ormore binding sites for a target associated with a disease or condition,such as a tumor-associated or autoimmune disease-associated antigen oran antigen produced or displayed by a pathogenic organism, such as avirus, bacterium, fungus or other microorganism. A second binding sitewill specifically bind to the targetable construct. Methods forpre-targeting using bispecific or multispecific antibodies are wellknown in the art (see, e.g., U.S. Pat. No. 6,962,702, the entirecontents of which are incorporated herein by reference.) Similarly,antibodies or fragments thereof that bind to targetable constructs arealso well known in the art (Id.), such as the 679 monoclonal antibodythat binds to HSG (histamine succinyl glycine). Generally, inpretargeting methods the bispecific or multispecific antibody isadministered first and allowed to bind to cell or tissue targetantigens. After an appropriate amount of time for unbound antibody toclear from circulation, the e.g. F-18 labeled targetable construct isadministered to the patient and binds to the antibody localized totarget cells or tissues, then an image is taken for example by PETscanning.

In an exemplary embodiment, a non-peptide receptor targeting agent suchas folic acid may be conjugated to NOTA and then labeled with, forexample, an F-18 metal complex that binds to NOTA. Such non-peptidereceptor targeting agents may include, for example, TA138, a non-peptideantagonist for the integrin α_(v)β₃ receptor (Liu et al., 2003, Bioconj.Chem. 14:1052-56). Similar non-peptide targeting agents known in the artthat can be conjugated to DOTA, NOTA or another chelating agent for F-18metal complexes may be utilized in the claimed methods. Other receptortargeting agents are known in the art, such as the somatostatin receptortargeting agent In-DTPA octreotide (TYCO®). As discussed below, anF-18-metal complex could potentially be chelated using DTPA and used forimaging purposes. The NODAGATOC peptide could be labeled with AlF-18 forsomatostatin receptor targeting (Eisenwiener et. al. Bioconj. Chem.2002, 13 (3):530-41). Other methods of receptor targeting imaging usingmetal chelates are known in the art and may be utilized in the practiceof the claimed methods (see, e.g., Andre et al., 2002, J. Inorg.Biochem. 88:1-6; Pearson et al., 1996, J. Med., Chem. 39:1361-71).

Imaging techniques and apparatus for F-18 imaging by PET scanning arealso well known in the art (see, e.g., U.S. Pat. Nos. 6,358,489;6,953,567; Page et al., Nuclear Medicine And Biology, 21:911-919, 1994;Choi et al., Cancer Research 55:5323-5329, 1995; Zalutsky et al., J.Nuclear Med., 33:575-582, 1992) and any such known PET imaging techniqueor apparatus may be utilized.

Although the Examples below demonstrate the use of F-18 metal complexesfor PET imaging, the skilled artisan will realize that stablemetal-fluorine complexes, such as the non- radioactive Al-27 and F-19complex, could also be bound to NOTA or other chelators and attached topeptides or other targeting agents for use as an MRI contrast agent. TheAlF NOTA complexes could also be attached to polymers for MRI imaging.The AlF NOTA derivatives could be used as PARACEST MRI imaging agents(Woessner et. al. Magn. Reson. Med. 2005, 53: 790-99).

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are included to illustrate particular embodimentsof the invention and are not meant to be limiting as to the scope of theclaimed subject matter.

FIG. 1. Exemplary peptide IMP 272.

FIG. 2. Exemplary peptide IMP 288.

FIG. 3. Exemplary peptide IMP 326.

FIG. 4. Exemplary peptide IMP 329.

FIG. 5. Exemplary peptide IMP 331.

FIG. 6. Exemplary peptide IMP 332.

FIG. 7. Exemplary peptide IMP 333.

FIG. 8. Exemplary peptide IMP 334.

FIG. 9. Exemplary peptide IMP 349.

FIG. 10. Exemplary peptide IMP 368.

FIG. 11. Exemplary peptide IMP 375.

FIG. 12. Exemplary peptide IMP 384.

FIG. 13. Exemplary peptide IMP 386.

FIG. 14. Exemplary peptide IMP 389.

FIG. 15. Exemplary peptide IMP 449.

FIG. 16. Additional exemplary peptides IMP 422, IMP 426 and IMP 428.

FIG. 17. Exemplary NOTA derivative.

FIG. 18. Exemplary NODA-peptide structure.

FIG. 19. Comparative biodistribution of In-111 and F-18 labeled IMP 449in mice with or without TF2 bispecific antibody.

FIG. 20. In vivo imaging of tumors using an ¹¹¹In-labeled diHSG peptide(IMP 288) with or without pretargeting TF 1O bispecific anti-MUC 1antibody.

FIG. 21. PET imaging of micrometastatic human colon cancer in lungs ofnude mice, using ¹²⁴I-labeled peptide and pretargeting with TF2bispecific anti-CEA antibody.

FIG. 22A-22D. Additional exemplary chelating moieties for use with F-18labeling.

DETAILED DESCRIPTION

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thedisclosure herein. Terms that are not explicitly defined are usedaccording to their plain and ordinary meaning.

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

As used herein, “about” means within plus or minus ten percent of anumber. For example, “about 100” would refer to any number between 90and 110.

As used herein, a “peptide” refers to any sequence of naturallyoccurring or non-naturally occurring amino acids of between 2 and 100amino acid residues in length, more preferably between 2 and 10, morepreferably between 2 and 6 amino acids in length. An “amino acid” may bean L-amino acid, a D-amino acid, an amino acid analogue, an amino acidderivative or an amino acid mimetic.

As used herein, a labeled molecule is “purified” when the labeledmolecule is partially or wholly separated from unlabeled molecules, sothat the fraction of labeled molecules is enriched compared to thestarting mixture. A “purified” labeled molecule may comprise a mixtureof labeled and unlabeled molecules in almost any ratio, including butnot limited to about 5:95; 10:90; 15:85; 20:80; 25:75; 30:70; 40:60;50:50; 60:40; 70:30; 75:25; 80:20; 85:15; 90:10; 95:5; 97:3; 98:2; 99:1or 100:0.

As used herein, the term “pathogen” includes, but is not limited tofungi, viruses, parasites and bacteria, including but not limited tohuman immunodeficiency virus (HIV), herpes virus, cytomegalovirus,rabies virus, influenza virus, hepatitis B virus, Sendai virus, felineleukemia virus, Reo virus, polio virus, human serum parvo-like virus,simian virus 40, respiratory syncytial virus, mouse mammary tumor virus,Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murineleukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Streptococcus agalactiae, Legionella pneumophilia, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, Mycobacterium tuberculosis and Chlostridium tetani.

As used herein, a “radiolysis protection agent” refers to any molecule,compound or composition that may be added to an F-18 labeled complex ormolecule to decrease the rate of breakdown of the F-18 labeled complexor molecule by radiolysis. Any known radiolysis protection agent,including but not limited to ascorbic acid, may be used.

Targetable Construct Peptides

In certain embodiments, the F-18 labeled moiety may comprise a peptideor other targetable construct. F-18 labeled peptides (or proteins) maybe selected to bind directly to a targeted cell, tissue, pathogenicorganism or other target for imaging and/or detection. In otherembodiments, F-18 labeled peptides may be selected to bind indirectly,for example using a bispecific antibody with one or more binding sitesfor a targetable construct peptide and one or more binding sites for atarget antigen associated with a disease or condition. Bispecificantibodies may be used, for example, in a pretargeting technique whereinthe antibody may be administered first to a subject. Sufficient time maybe allowed for the bispecific antibody to bind to a target antigen andfor unbound antibody to clear from circulation. Then a targetableconstruct, such as an F-18 labeled peptide, may be administered to thesubject and allowed to bind to the bispecific antibody and localize tothe diseased cell or tissue, after which the distribution of the F-18labeled targetable construct may be determined by PET scanning or otherknown techniques.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic. Aside frompeptides, carbohydrates may also be used.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons, including the metal ions in the chelates. More usually, thetargetable construct peptide will have four or more residues, such asthe peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (SEQ ID NO: 1). whereinDOTA is 1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is thehistamine succinyl glycyl group. Alternatively, the DOTA may be replacedby a NOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid) or TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) moiety.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids andpeptoids.

The peptides used as targetable constructs are synthesized convenientlyon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later for chelateconjugation, are advantageously blocked with standard protecting groupssuch as a Boc group, while N-terminal residues may be acetylated toincrease serum stability. Such protecting groups will be known to theskilled artisan. See Greene and Wuts Protective Groups in OrganicSynthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides areprepared for later use within the bispecific antibody system, they areadvantageously cleaved from the resins to generate the correspondingC-terminal amides, in order to inhibit in vivo carboxypeptidaseactivity.

The haptens of the immunogen comprise a recognition moiety, for example,a chemical hapten. Using a chemical hapten, preferably the HSG hapten,high specificity of the linker for the antibody is exhibited. Antibodiesraised to the HSG hapten are known and can be easily incorporated intothe appropriate bispecific antibody (see, e.g., U.S. Pat. Nos.6,962,702; 7,138,103 and 7,300,644, the entire text of each of which isincorporated herein by reference). Thus, binding of the linker with theattached hapten would be highly specific for the antibody or antibodyfragment.

Chelate Moieties

In some embodiments, an F-18 labeled molecule may comprise one or morehydrophilic chelate moieties, which can bind metal ions and also help toensure rapid in vivo clearance. Chelators may be selected for theirparticular metal-binding properties, and may be readily interchanged.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs. Macrocyclic chelators such asNOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid), DOTA, and TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are also ofuse with a variety of metals, that may potentially be used as ligandsfor F-18 conjugation.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers are of interest forstably binding nuclides. Porphyrin chelators may be used with numerousmetal complexes. More than one type of chelator may be conjugated to acarrier to bind multiple metal ions. Chelators such as those disclosedin U.S. Pat. No. 5,753,206, especially thiosemicarbazonylglyoxylcysteine(Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelatorsare advantageously used to bind soft acid cations of Tc, Re, Bi andother transition metals, lanthanides and actinides that are tightlybound to soft base ligands. It can be useful to link more than one typeof chelator to a peptide. Because antibodies to a di-DTPA hapten areknown (Barbet et al., U.S. Pat. No. 5,256,395) and are readily coupledto a targeting antibody to form a bispecific antibody, it is possible touse a peptide hapten with cold diDTPA chelator and another chelator forbinding an F-18 complex, in a pretargeting protocol. One example of sucha peptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH₂ (SEQ ID NO:2).Other hard acid chelators such as DOTA, TETA and the like can besubstituted for the DTPA and/or Tscg-Cys groups, and MAbs specific tothem can be produced using analogous techniques to those used togenerate the anti-di-DTPA MAb.

Another useful chelator may comprise a NOTA-type moiety, for example asdisclosed in Chong et al. (Rational design and generation of a bimodalbifunctional ligand for antibody-targeted radiation cancer therapy, J.Med. Chem., e-published on Dec. 7, 2007, incorporated herein byreference). Chong et al. disclose the production and use of abifunctional C-NETA ligand, based upon the NOTA structure, that whencomplexed with ¹⁷⁷Lu or ^(205/206)Bi showed stability in serum for up to14 days.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the targetable construct, e.g., withdifferent chelate ring sizes, to bind preferentially to two differenthard acid or soft acid cations, due to the differing sizes of thecations, the geometries of the chelate rings and the preferred complexion structures of the cations. This will permit two different metals,one or both of which may be attached to F-18, to be incorporated into atargetable construct for eventual capture by a pretargeted bispecificantibody.

Methods of Administration

In various embodiments, bispecific antibodies and targetable constructsmay be used for imaging normal or diseased tissue and organs (see, e.g.U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094;5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679;5,128,119; 5,101,827; and 4,735,210, each incorporated herein byreference in its entirety).

The administration of a bispecific antibody (bsAb) and an F-18 labeledtargetable construct may be conducted by administering the bsAb antibodyat some time prior to administration of the targetable construct. Thedoses and timing of the reagents can be readily devised by a skilledartisan, and are dependent on the specific nature of the reagentsemployed. If a bsAb-F(ab′)₂ derivative is given first, then a waitingtime of 24-72 hr (alternatively 48-96 hours) before administration ofthe targetable construct would be appropriate. If an IgG-Fab′ bsAbconjugate is the primary targeting vector, then a longer waiting periodbefore administration of the targetable construct would be indicated, inthe range of 3-10 days. After sufficient time has passed for the bsAb totarget to the diseased tissue, the F-18 labeled targetable construct isadministered. Subsequent to administration of the targetable construct,imaging can be performed.

Certain embodiments concern the use of multivalent target bindingproteins which have at least three different target binding sites asdescribed in patent application Ser. No. 60/220,782. Multivalent targetbinding proteins have been made by cross-linking several Fab-likefragments via chemical linkers. See U.S. Pat. Nos. 5,262,524; 5,091,542and Landsdorp et al. Euro. J. Immunol. 16: 679-83 (1986). Multivalenttarget binding proteins also have been made by covalently linkingseveral single chain Fv molecules (scFv) to form a single polypeptide.See U.S. Pat. No. 5,892,020. A multivalent target binding protein whichis basically an aggregate of scFv molecules has been disclosed in U.S.Pat. Nos. 6,025,165 and 5,837,242. A trivalent target binding proteincomprising three scFv molecules has been described in Krott et al.Protein Engineering 10(4): 423-433 (1997).

Alternatively, a technique known as “dock-and-lock” (DNL) has beendemonstrated for the simple and reproducible construction of a varietyof multivalent complexes, including complexes comprising two or moredifferent antibodies or antibody fragments. (See, e.g., U.S. patentapplication Ser. Nos. 11/389,358, filed Mar. 24, 2006; Ser. No.11/391,584, filed Mar. 28, 2006; Ser. No. 11/478,021, filed Jun. 29,2006; Ser. No. 11/633,729, filed Dec. 5, 2006; and Ser. No. 11/925,408,filed Oct. 26, 2007, the text of each of which is incorporated herein byreference in its entirety.) Such constructs are also of use for thepractice of the claimed methods and compositions described herein.

A clearing agent may be used which is given between doses of thebispecific antibody (bsAb) and the targetable construct. A clearingagent of novel mechanistic action may be used, namely a glycosylatedanti-idiotypic Fab′ fragment targeted against the disease targetingarm(s) of the bsAb. In one example, anti-CEA (MN-14 Ab) x anti-peptidebsAb is given and allowed to accrete in disease targets to its maximumextent. To clear residual bsAb, an anti-idiotypic Ab to MN-14, termedW12, is given, preferably as a glycosylated Fab′ fragment. The clearingagent binds to the bsAb in a monovalent manner, while its appendedglycosyl residues direct the entire complex to the liver, where rapidmetabolism takes place. Then the F-18 labeled targetable construct isgiven to the subject. The W12 Ab to the MN-14 arm of the bsAb has a highaffinity and the clearance mechanism differs from other disclosedmechanisms (see Goodwin et al., ibid), as it does not involvecross-linking, because the W12-Fab′ is a monovalent moiety. However,alternative methods and compositions for clearing agents are known andany such known clearing agents may be used.

Formulation and Administration

The F-18 labeled molecules may be formulated to obtain compositions thatinclude one or more pharmaceutically suitable excipients, one or moreadditional ingredients, or some combination of these. These can beaccomplished by known methods to prepare pharmaceutically usefuldosages, whereby the active ingredients (i.e., the F-18 labeledmolecules) are combined in a mixture with one or more pharmaceuticallysuitable excipients. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell known to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The preferred route for administration of the compositions describedherein is parental injection. Injection may be intravenous,intraarterial, intralymphatic, intrathecal, or intracavitary (i.e.,parenterally). In parenteral administration, the compositions will beformulated in a unit dosage injectable form such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives. Other methodsof administration, including oral administration, are also contemplated.

Formulated compositions comprising F-18 labeled molecules can be usedfor intravenous administration via, for example, bolus injection orcontinuous infusion. Compositions for injection can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. Compositions can also take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and cancontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the compositions can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The compositions may be administered in solution. The pH of the solutionshould be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. Theformulation thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate, TRIS(hydroxymethyl) aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The formulatedsolution may also contain a salt, such as sodium chloride or potassiumchloride in a concentration of 50 to 150 mM. An effective amount of astabilizing agent such as glycerol, albumin, a globulin, a detergent, agelatin, a protamine or a salt of protamine may also be included. Thecompositions may be administered to a mammal subcutaneously,intravenously, intramuscularly or by other parenteral routes. Moreover,the administration may be by continuous infusion or by single ormultiple boluses.

Where bispecific antibodies are administered, for example in apretargeting technique, the dosage of an administered antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, for imaging purposes it is desirable to provide therecipient with a dosage of bispecific antibody hat is in the range offrom about 1 mg to 200 mg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. Typically, it is desirable to provide the recipient with adosage that is in the range of from about 10 mg per square meter of bodysurface area or 17 to 18 mg of the antibody for the typical adult,although a lower or higher dosage also may be administered ascircumstances dictate. Examples of dosages of bispecific antibodies thatmay be administered to a human subject for imaging purposes are 1 to 200mg, more preferably 1 to 70 mg, most preferably 1 to 20 mg, althoughhigher or lower doses may be used.

In general, the dosage of F-18 label to administer will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Preferably, a saturatingdose of the F-18 labeled molecules is administered to a patient. Foradministration of F-18 labeled molecules, the dosage may be measured bymillicuries. A typical range for F-18 imaging studies would be five to10 mCi.

Administration of Peptides

Various embodiments of the claimed methods and/or compositions mayconcern one or more F-18 labeled peptides to be administered to asubject. Administration may occur by any route known in the art,including but not limited to oral, nasal, buccal, inhalational, rectal,vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial, intrathecal or intravenous injection.Where, for example, F-18 labeled peptides are administered in apretargeting protocol, the peptides would preferably be administeredi.v.

Unmodified peptides administered orally to a subject can be degraded inthe digestive tract and depending on sequence and structure may exhibitpoor absorption across the intestinal lining. However, methods forchemically modifying peptides to render them less susceptible todegradation by endogenous proteases or more absorbable through thealimentary tract are well known (see, for example, Blondelle et al.,1995, Biophys. J. 69:604-11; Ecker and Crooke, 1995, Biotechnology13:351-69; Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMISTRY AND DRUGDISCOVERY, VOL. I, ed. Wollf, John Wiley & Sons; Goodman and Shao, 1996,Pure & Appl. Chem. 68:1303-08). Methods for preparing libraries ofpeptide analogs, such as peptides containing D-amino acids;peptidomimetics consisting of organic molecules that mimic the structureof a peptide; or peptoids such as vinylogous peptoids, have also beendescribed and may be used to construct peptide based F-18 labeledmolecules suitable for oral administration to a subject.

In certain embodiments, the standard peptide bond linkage may bereplaced by one or more alternative linking groups, such as CH₂—NH,CH₂—S, CH₂—CH₂, CH═CH, CO—CH₂, CHOH—CH₂ and the like. Methods forpreparing peptide mimetics are well known (for example, Hruby, 1982,Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest etal., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int. J. Pept.Res. 14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Pat.Nos. 5,169,862; 5,539,085; 5,576,423, 5,051,448, 5,559,103, eachincorporated herein by reference.) Peptide mimetics may exhibit enhancedstability and/or absorption in vivo compared to their peptide analogs.

Alternatively, peptides may be administered by oral delivery usingN-terminal and/or C-terminal capping to prevent exopeptidase activity.For example, the C-terminus may be capped using amide peptides and theN-terminus may be capped by acetylation of the peptide. Peptides mayalso be cyclized to block exopeptidases, for example by formation ofcyclic amides, disulfides, ethers, sulfides and the like.

Peptide stabilization may also occur by substitution of D-amino acidsfor naturally occurring L-amino acids, particularly at locations whereendopeptidases are known to act. Endopeptidase binding and cleavagesequences are known in the art and methods for making and using peptidesincorporating D-amino acids have been described (e.g., U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Jun. 14,2004, incorporated herein by reference). In certain embodiments,peptides and/or proteins may be orally administered by co-formulationwith proteinase- and/or peptidase-inhibitors.

Other methods for oral delivery of therapeutic peptides are disclosed inMehta (“Oral delivery and recombinant production of peptide hormones,”June 2004, BioPharm International). The peptides are administered in anenteric-coated solid dosage form with excipients that modulateintestinal proteolytic activity and enhance peptide transport across theintestinal wall. Relative bioavailability of intact peptides using thistechnique ranged from 1% to 10% of the administered dosage. Insulin hasbeen successfully administered in dogs using enteric-coatedmicrocapsules with sodium cholate and a protease inhibitor (Ziv et al.,1994, J. Bone Miner. Res. 18 (Suppl. 2):792-94. Oral administration ofpeptides has been performed using acylcarnitine as a permeation enhancerand an enteric coating (Eudragit L30D-55, Rohm Pharma Polymers, seeMehta, 2004). Excipients of use for orally administered peptides maygenerally include one or more inhibitors of intestinalproteases/peptidases along with detergents or other agents to improvesolubility or absorption of the peptide, which may be packaged within anenteric-coated capsule or tablet (Mehta, 2004). Organic acids may beincluded in the capsule to acidify the intestine and inhibit intestinalprotease activity once the capsule dissolves in the intestine (Mehta,2004). Another alternative for oral delivery of peptides would includeconjugation to polyethylene glycol (PEG)-based amphiphilic oligomers,increasing absorption and resistance to enzymatic degradation (Solteroand Ekwuribe, 2001, Pharm. Technol. 6:110).

Methods for Raising Antibodies

Abs to peptide backbones may be generated by well-known methods for Abproduction. For example, injection of an immunogen, such as(peptide)_(n)-KLH, wherein KLH is keyhole limpet hemocyanin, and n-1-30,in complete Freund's adjuvant, followed by two subsequent injections ofthe same immunogen suspended in incomplete Freund's adjuvant intoimmunocompetent animals, is followed three days after an i.v. boost ofantigen, by spleen cell harvesting. Harvested spleen cells are thenfused with Sp2/0-Ag14 myeloma cells and culture supernatants of theresulting clones analyzed for anti-peptide reactivity using adirect-binding ELISA. Specificity of generated Abs can be analyzed forby using peptide fragments of the original immunogen. These fragmentscan be prepared readily using an automated peptide synthesizer. For Abproduction, enzyme-deficient hybridomas are isolated to enable selectionof fused cell lines. This technique also can be used to raise antibodiesto one or more of the chelates comprising the targetable construct,e.g., In(III)-DTPA chelates. Monoclonal mouse antibodies to anIn(III)-di-DTPA are known (Barbet '395 supra).

Targeting antibodies of use, for example as components of bispecificantibodies, may be specific to a variety of cell surface orintracellular tumor-associated antigens as marker substances. Thesemarkers may be substances produced by the tumor or may be substanceswhich accumulate at a tumor site, on tumor cell surfaces or within tumorcells, whether in the cytoplasm, the nucleus or in various organelles orsub-cellular structures. Among such tumor-associated markers are thosedisclosed by Herberman, “Immunodiagnosis of Cancer”, in Fleisher ed.,“The Clinical Biochemistry of Cancer”, page 347 (American Association ofClinical Chemists, 1979) and in U.S. Pat. Nos. 4,150,149; 4,361,544; and4,444,744, each incorporated herein by reference. Recent reports ontumor associated antigens include Mizukami et al., (2005, Nature Med.11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets 5:229-48);Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Ren et al.(2005, Ann. Surg. 242:55-63), each incorporated herein by reference.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

Another marker of interest is transmembrane activator andCAML-interactor (TACI). See Yu et al. Nat. Immunol. 1:252-256 (2000).Briefly, TACI is a marker for B-cell malignancies (e.g., lymphoma).Further it is known that TACI and B cell maturation antigen (BCMA) arebound by the tumor necrosis factor homolog—a proliferation-inducingligand (APRIL). APRIL stimulates in vitro proliferation of primary B andT cells and increases spleen weight due to accumulation of B cells invivo. APRIL also competes with TALL-I (also called BLyS or BAFF) forreceptor binding. Soluble BCMA and TACI specifically prevent binding ofAPRIL and block APRIL-stimulated proliferation of primary B cells.BCMA-Fc also inhibits production of antibodies against keyhole limpethemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-Isignaling via BCMA and/or TACI are required for generation of humoralimmunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligand-tworeceptor pathway involved in stimulation of B and T cell function.

Exemplary target antigens of use for imaging various diseases orconditions, such as a malignant disease, a cardiovascular disease, aninfectious disease, an inflammatory disease, an autoimmune disease, or aneurological disease may include colon-specific antigen-p (CSAp),carcinoembryonic antigen (CEA), CD4, CD5, CD8, CD14, CD15, CD19, CD20,CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, Ia, Ii,MUC 1, MUC 2, MUC 3, MUC 4, NCA (CEACAM6 or CD66a-d and CD67, as well asCD138), EGFR, HER 2/neu, TAG-72, EGP-1, EGP-2, A3, KS-1, Le(y), S100,PSMA, PSA, tenascin, folate receptor, VEGFR, PlGF, ILGF-1, necrosisantigens, IL-2, IL-6, T101, MAGE, or a combination of these antigens. Inparticular, antigens may include carcinoembryonic antigen (CEA),tenascin, epidermal growth factor receptor, platelet derived growthfactor receptor, fibroblast growth factor receptors, vascularendothelial growth factor receptors, gangliosides, HER/2neu receptorsand combinations of these antigens.

Where imaging or detection involves a lymphoma, leukemia or autoimmunedisorder, targeted antigens may be selected from the group consisting ofCD4, CD5, CD8, CD 14, CD 15, CD19, CD20, CD21, CD22, CD23, CD25, CD33,CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80,CD126, CD138, CD154, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF,PlGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d,necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) andTRAIL-R2 (DR5).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by the publication of Orlandi et al., Proc. Nat'l Acad. Sci.USA 86: 3833 (1989), which is incorporated by reference in its entirety.Techniques for producing humanized MAbs are described, for example, byJones et al., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323(1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc.Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J. Immun. 150: 2844 (1993), each of whichis incorporated herein by reference in its entirety.

Alternatively, fully human antibodies can be obtained from transgenicnon-human animals. See, e.g., Mendez et al., Nature Genetics, 15:146-156 (1997); U.S. Pat. No. 5,633,425. For example, human antibodiescan be recovered from transgenic mice possessing human immunoglobulinloci. The mouse humoral immune system is humanized by inactivating theendogenous immunoglobulin genes and introducing human immunoglobulinloci. The human immunoglobulin loci are exceedingly complex and comprisea large number of discrete segments which together occupy almost 0.2% ofthe human genome. To ensure that transgenic mice are capable ofproducing adequate repertoires of antibodies, large portions of humanheavy- and light-chain loci must be introduced into the mouse genome.This is accomplished in a stepwise process beginning with the formationof yeast artificial chromosomes (YACs) containing either human heavy- orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the YAC through their germline and are bred with mice deficientin murine antibody production. Breeding the two transgenic strains, onecontaining the human heavy-chain loci and the other containing the humanlight-chain loci, creates progeny which produce human antibodies inresponse to immunization.

Unrearranged human immunoglobulin genes also can be introduced intomouse embryonic stem cells via microcell-mediated chromosome transfer(MMCT). See, e.g., Tomizuka et al., Nature Genetics, 16: 133 (1997). Inthis methodology microcells containing human chromosomes are fused withmouse embryonic stem cells. Transferred chromosomes are stably retained,and adult chimeras exhibit proper tissue-specific expression.

As an alternative, an antibody or antibody fragment may be derived fromhuman antibody fragments isolated from a combinatorial immunoglobulinlibrary. See, e.g., Barbas et al., METHODS: A Companion to Methods inEnzymology 2: 119 (1991), and Winter et al., Ann. Rev. Immunol. 12: 433(1994), which are incorporated herein by reference. Many of thedifficulties associated with generating monoclonal antibodies by B-cellimmortalization can be overcome by engineering and expressing antibodyfragments in E. coli, using phage display.

A similar strategy can be employed to obtain high-affinity scFv. See,e.g., Vaughn et al., Nat. Biotechnol., 14: 309-314 (1996). An scFvlibrary with a large repertoire can be constructed by isolating V-genesfrom non-immunized human donors using PCR primers corresponding to allknown V_(H), V_(kappa) and V₈₀ gene families. Following amplification,the V_(kappa) and V_(lambda) pools are combined to form one pool. Thesefragments are ligated into a phagemid vector. The scFv linker, (Gly₄,Ser)₃, is then ligated into the phagemid upstream of the V_(L) fragment.The V_(H) and linker-V_(L) fragments are amplified and assembled on theJ_(H) region. The resulting V_(H)-linker-V_(L) fragments are ligatedinto a phagemid vector. The phagemid library can be panned usingfilters, as described above, or using immunotubes (NUNC®; MAXISORP®).Similar results can be achieved by constructing a combinatorialimmunoglobulin library from lymphocytes or spleen cells of immunizedrabbits and by expressing the scFv constructs in P. pastoris. See, e.g.,Ridder et al., Biotechnology, 13: 255-260 (1995). Additionally,following isolation of an appropriate scFv, antibody fragments withhigher binding affinities and slower dissociation rates can be obtainedthrough affinity maturation processes such as CDR3 mutagenesis and chainshuffling. See, e.g., Jackson et al., Br. J. Cancer, 78: 181-188 (1998);Osbourn et al., Immunotechnology, 2: 181-196 (1996).

Another form of an antibody fragment is a peptide coding for a singleCDR. CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106 (1991); Courtenay-Luck, “Genetic Manipulation ofMonoclonal Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages166-179 (Cambridge University Press 1995); and Ward et al., “GeneticManipulation and Expression of Antibodies,” in MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages 137-185(Wiley-Liss, Inc. 1995).

Bispecific antibodies can be prepared by techniques known in the art,for example, an anti-CEA tumor Ab and an anti-peptide Ab are bothseparately digested with pepsin to their respective F(ab′)₂ fragments.The anti-CEA-Ab-F(ab′)₂ is reduced with cysteine to generate Fab′monomeric units which are further reacted with the cross-linkerbis(maleimido) hexane to produce Fab′-maleimide moieties. Theanti-peptide Ab-F(ab′)₂ is reduced with cysteine and the purified,recovered anti-peptide Fab′-SH is reacted with theanti-CEA-Fab′-maleimide to generate the Fab′×Fab′ bi-specific Ab.Alternatively, the anti-peptide Fab′-SH fragment may be coupled with theanti-CEA F(ab′)₂ to generate a F(ab′)₂×Fab′ construct, or with anti-CEAIgG to generate an IgG×Fab′ bi-specific construct. In one embodiment,the IgG×Fab′ construct can be prepared in a site-specific manner byattaching the antipeptide Fab′ thiol group to anti-CEA IgG heavy-chaincarbohydrate which has been periodate-oxidized, and subsequentlyactivated by reaction with a commercially available hydrazide-maleimidecross-linker. The component Abs used can be chimerized or humanized byknown techniques. A chimeric antibody is a recombinant protein thatcontains the variable domains and complementary determining regionsderived from a rodent antibody, while the remainder of the antibodymolecule is derived from a human antibody. Humanized antibodies arerecombinant proteins in which murine complementarity determining regionsof a monoclonal antibody have been transferred from heavy and lightvariable chains of the murine immunoglobulin into a human variabledomain.

A chimeric Ab is constructed by ligating the cDNA fragment encoding themouse light variable and heavy variable domains to fragment encoding theC domains from a human antibody. Because the C domains do not contributeto antigen binding, the chimeric antibody will retain the same antigenspecificity as the original mouse Ab but will be closer to humanantibodies in sequence. Chimeric Abs still contain some mouse sequences,however, and may still be immunogenic. A humanized Ab contains onlythose mouse amino acids necessary to recognize the antigen. This productis constructed by building into a human antibody framework the aminoacids from mouse complementarity determining regions.

Other recent methods for producing bispecific antibodies includeengineered recombinant Abs which have additional cysteine residues sothat they crosslink more strongly than the more common immunoglobulinisotypes. See, e.g., FitzGerald et al., Protein Eng. 10:1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bi-specific fusion proteins canbe produced using molecular engineering. In one form, the bi-specificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bi-specificfusion protein is divalent, consisting of, for example, an IgG with twobinding sites for one antigen and two scFv with two binding sites for asecond antigen.

Functional bi-specific single-chain antibodies (bscAb), also calleddiabodies, can be produced in mammalian cells using recombinant methods.See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995.

Preferred bispecific antibodies are those which incorporate the Fv ofMAb Mu-9 and the Fv of MAb 679 or the Fv of MAb MN-14 and the Fv of MAb679, and their human, chimerized or humanized counterparts. The MN-14,as well as its chimerized and humanized counterparts, are disclosed inU.S. Pat. No. 5,874,540. Also preferred are bispecific antibodies whichincorporate one or more of the CDRs of Mu-9 or 679. The antibody canalso be a fusion protein or a bispecific antibody that incorporates aClass III anti-CEA antibody and the Fv of 679. Class III antibodies,including Class III anti-CEA are discussed in detail in U.S. Pat. No.4,818,709.

The skilled artisan will realize that bispecific antibodies mayincorporate any antibody or fragment known in the art that has bindingspecificity for a target antigen that is known to be associated with adisease state or condition. Such known antibodies include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), hA20 (anti-CD20),RS7 (anti-epithelial glycoprotein-1 (EGP-1)), PAM-4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e)), MN-3 or MN-15 (NCA or CEACAM6), Mu-9 (anti-colon-specificantigen-p), Immu 31 (an anti-alpha-fetoprotein), TAG-72 (e.g., CC49),Tn, J591 (anti-PSMA (prostate-specific membrane antigen)), G250 (ananti-carbonic anhydrase IX MAb) and L243 (anti-HLA-DR). Such antibodiesare known in the art (e.g., U.S. Pat. Nos. 5,686,072; 5,874,540;6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730,300; 6,899,864;6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786;7,256,004; 7,282,567; 7,300,655; 7,312,318; and U.S. Patent ApplicationPubl. No. 20040185053; 20040202666; 20050271671; 20060193865;20060210475; 20070087001; each incorporated herein by reference in itsentirety.) Such known antibodies are of use for detection and/or imagingof a variety of disease states or conditions (e.g., hMN-14 or TF2 bsMAb(CEA-expressing carcinomas), hA20 bsMab (TF-4-lymphoma), hPAM4 (TF-10pancreas cancers), RS7 bsMAb (lung, breast, ovarian, prostatic cancers),hMN-15 or hMN3 bsMAb (inflammation), human gp120 and/or gp41 bsMAbs(HIV), anti-platelet bsMab and anti-thrombin bsMAb (clot imaging),anti-myosin bsMAb (cardiac necrosis)).

Candidate anti-HIV antibodies include the anti-envelope antibodydescribed by Johansson et al. (AIDS. 2006 Oct. 3; 20 (15):1911-5), aswell as the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21 (16):2161-2170 and Joos etal., Antimicrob. Agens Chemother. 2006; 50 (5): 1773-9, all incorporatedherein in their entirety by reference.

In certain embodiments, the bsAb F-18 labeled targetable constructsdiscussed above may be used in intraoperative, intravascular, and/orendoscopic tumor and lesion detection, biopsy and therapy as describedin U.S. Pat. No. 6,096,289.

Imaging Using Labeled Molecules

Methods of imaging using labeled molecules are well known in the art,and any such known methods may be used with the fluoride-labeledmolecules disclosed herein. See, e.g., U.S. Pat. Nos. 6,241,964;6,358,489; 6,953,567 and published U.S. Patent Application Publ. Nos.20050003403; 20040018557; 20060140936, each incorporated herein byreference in its entirety. See also, Page et al., Nuclear Medicine AndBiology, 21:911-919, 1994; Choi et al., Cancer Research 55:5323-5329,1995; Zalutsky et al., J. Nuclear Med., 33:575-582, 1992; Woessner et.al. Magn. Reson. Med. 2005, 53: 790-99.

In certain embodiments, F-18 labeled molecules may be of use in imagingnormal or diseased tissue and organs, for example using the methodsdescribed in U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095;5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902;5,328,679; 5,128,119; 5,101,827; and 4,735,210, each incorporated hereinby reference. Additional methods are described in U.S. application Ser.No. 09/337,756 filed Jun. 22, 1999 and in U.S. application Ser. No.09/823,746, filed Apr. 3, 2001. Such imaging can be conducted by directF-18 labeling of the appropriate targeting molecules, or by apretargeted imaging method, as described in Goldenberg et al. (2007,Update Cancer Ther. 2:19-31); Sharkey et al. (2008, Radiology246:497-507); Goldenberg et al. (2008, J. Nucl. Med. 49:158-63); Sharkeyet al. (2007, Clin. Cancer Res. 13:5777s-5585s); McBride et al. (2006,J. Nucl. Med. 47:1678-88); Goldenberg et al. (2006, J. Clin.Oncol.24:823-85), see also U.S. Patent Publication Nos. 20050002945,20040018557, 20030148409 and 20050014207, each incorporated herein byreference.

Methods of diagnostic imaging with labeled peptides or MAbs arewell-known. For example, in the technique of immunoscintigraphy, ligandsor antibodies are labeled with a gamma-emitting radioisotope andintroduced into a patient. A gamma camera is used to detect the locationand distribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Alsopreferred is the use of positron-emitting radionuclides (PET isotopes),such as with an energy of 511 keV, such as ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, and ¹²⁴I.Such radionuclides may be imaged by well-known PET scanning techniques.

EXAMPLES Example 1 F-18 Labeling of Peptide IMP 272

The first peptide that was used was IMP 272:

-   -   DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂ MH⁺1512

Acetate buffer solution—Acetic acid, 1.509 g was diluted in 160 mL waterand the pH was adjusted by the addition of 1 M NaOH then diluted to 250mL to afford a 0.1 M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAcsolution at pH 4 to provide a 2 mM Al stock solution.

IMP 272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP 272was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

F-18 Labeling of IMP 272—A 3 μL aliquot of the aluminum stock solutionwas placed in a REACTI-VIAL™ and mixed with 50 μL F-18 (as received) and3 μL of the IMP 272 solution. The solution was heated in a heating blockat 110° C. for 15 min and analyzed by reverse phase HPLC. The HPLC trace(not shown) showed 93% free F-18 and 7% bound to the peptide. Anadditional 10 μL of the IMP 272 solution was added to the reaction andit was heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% F-18 at the void volume and 92% of the activityattached to the peptide. The remainder of the peptide solution wasincubated at room temperature with 150μL PBS for ˜1 hr and then examinedby reverse phase HPLC. The HPLC (not shown) showed 58% F-18 unbound and42% still attached to the peptide. The data indicate that F-18-Al-DTPAcomplex may be unstable when mixed with phosphate.

Reverse Phase HPLC—Reverse phase HPLC analysis was done under thefollowing conditions:

-   -   Column: WATERS® XTERRA™ MS C₁₈ 5 μm, 4.6×250 mm    -   Flow Rate: 1 mL/min    -   Gradient Buffers: Buffer C, 0.1% NH₄OAc in DI water, Buffer D,        90% acetonitrile 10% water and 0.1% NH₄OAc    -   Gradient: 100% Buffer C to 100% Buffer D using a linear gradient        over 30 min.    -   Run Time: 30 min

Size Exclusion HPLC—The size exclusion HPLC was done under the followingconditions:

-   -   Column: BIORAD® BIO-SIL™SEC 250, 300×7.8 mm    -   Gradient: Isocratic    -   Eluent Buffer: 0.2 M Phosphate pH 6.8    -   Flow Rate: 1 mL/min    -   Run Time: 30 min

All radiometric traces were obtained using a PERKIN ELMER® 610Tr tomonitor the emission of F-18. Tables 1-3 are tabular representations ofthe data.

TABLE 1 F-18 + IMP 272 + AlCl₃ heated at 110° C. for 15 min, followed byanalysis by reverse phase HPLC. Regions: F-18 Detector: FSA Start EndRetention Height Area % % Total Name (mins) (mins) (mins) (CPM) (CPM)ROI (%) (%) Bkg 1 2.20 2.30 2.20 130.0 Region 1 2.30 3.30 2.60 85270.0200050.0 93.15 96.31 Bkg 2 4.40 4.50 4.40 210.0 Region 2 8.70 9.80 9.005590.0 14720.0 6.85 7.09 2 Peaks 214770.0 100.00 103.40

TABLE 2 F-18 + excess IMP 272 + AlCl₃ heated at 110° C. for 15 min,followed by analysis by reverse phase HPLC. Regions: F-18 Detector: FSAStart End Retention Height Area % % Total Name (mins) (mins) (mins)(CPM) (CPM) ROI (%) (%) Bkg 1 2.20 2.30 2.20 340.0 Region 1 2.40 3.202.70 6450.0 20549.6 7.76 8.23 Bkg 2 7.10 7.20 7.10 630.0 Region 2 7.308.70 8.50 3140.0 13113.6 4.95 5.25 Region 3 8.70 10.00 9.00 93700.0231023.9 87.28 92.57 Bkg 3 10.70 10.80 10.70 520.0 3 Peaks 264687.1100.00 106.06

TABLE 3 Phosphate Challenge in PBS for 90 min at room temp. Aliquot ofF-18 + excess IMP 272 + AlCl₃ heated at 110° C. for 15 min and analyzedby reverse phase HPLC. Regions: F-18 Detector: FSA Start End RetentionHeight Area % % Total Name (mins) (mins) (mins) (CPM) (CPM) ROI (%) (%)Bkg 1 2.00 2.10 2.00 350.0 Region 1 2.40 3.30 2.70 81930.0 162403.658.23 62.44 Bkg 2 4.20 4.30 4.20 410.0 Bkg 3 7.50 7.60 7.50 780.0 Region2 7.80 8.60 8.40 2110.0 5564.7 2.00 2.14 Region 3 8.60 9.80 8.90 44590.0110942.0 39.78 42.66 Bkg 4 10.50 10.60 10.50 460.0 3 Peaks 278910.3100.00 107.24

The labeled peptide was purified by applying the labeled peptidesolution onto a 1 cc (30 mg) WATERS® HLB column (Part # 186001879) andwashing with 300 μL water to remove unbound F-18. The peptide was elutedby washing the column with 2×100 μL 1:1 MeOH/H₂O. The purified peptidewas incubated in water at 25° C. and analyzed by reverse phase HPLC (notshown). The HPLC analysis showed that the F-18 labeled IMP 272 was notstable in water. After 40 min incubation in water about 17% of the F-18was released from the peptide, while 83% was retained (not shown).

Example 2 Immunoreactivity of F-18 IMP 272

The peptide (16 μL 2 mM IMP 272, 48 μg) was labeled with F-18 andanalyzed for antibody binding by size exclusion HPLC. The size exclusionHPLC showed that the peptide bound hMN-14 x 679 but did not bind to theirrelevant bispecific antibody hMN-14 x 734 (not shown).

Example 3 IMP 272 F-18 Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10⁻⁹ mol) was placed in apolypropylene cone vial and mixed with 75 μL F-18 (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁻⁸ mol) IMP 272 solution in 0.1 M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown).

Example 4 Standard F-18 Peptide Labeling Conditions Used to Screen OtherPeptides For Al-¹⁸F Binding

A 3 μL aliquot of the 2 mM aluminum stock solution was placed in apolypropylene cone vial and mixed with 50 μL F-18 (as received),incubated at room temperature for ˜2 min and then mixed with 16 to 20 μLof a 2 mM peptide solution in 0.1 M NaOAc pH 4 buffer. The solution washeated in a heating block at 100° C. for 15 min and analyzed by reversephase HPLC (PHENOMENEX™, GEMINI®, 5μ, C-18, 110A, 250×4.6 mm HPLCColumn).

Peptides Tested

IMP 272: DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂ MH⁺1512 (FIG. 1)

IMP 288 DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ MH⁺1453 (FIG. 2)

IMP 326 DTPA-ITC-NH-NH-Phe-CO-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂MH⁺1477 (FIG. 3)

IMP 329Deferoxamine-NH-CS-NH-NH-Ph-CO-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂MH⁺1804 (FIG. 4)

IMP 331 NTA-iAsp-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺1240 (FIG. 5)

IMP 332 EDTADpr-D-Ala-D-Lys(HSG)-D-Ala-D-Lsy(HSG)-NH₂ MH⁺1327 (FIG. 6)

IMP 333 DTPA-Dpr(DTPA)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1845(FIG. 7)

IMP 334 (H₂O₃P)₂—C(OH)—(CH₂)₃—NH-Gly-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂MH⁺1192 (FIG. 8)

IMP 337Ac-D-Ser(PO₃H₂)-D-Ser(PO₃H2)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1291

IMP 338 Ac-D-Ser(PO₃H₂)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1126

IMP 345 DTPA-D-Ser(PO₃H₂)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1459

IMP 349DTPA-D-Cys((H₂O₃P)₂—CH—CH₂—S)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1583 (FIG. 9)

IMP 361 DTPA-Dpr(BrCH₂CO—)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1498

IMP 366 DTPA-Dpr(Ph-S—CH₂CO—)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1528

IMP 368 Sym-DTPA-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1292 (FIG. 10)

IMP 369Sym-DTPA-NH—CH(2—Br-Phe-)-CH₂—CO-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1517

IMP 370Sym-DTPA-NH-CH(2—O₂N-Phe-)-CH₂—CO-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1484

IMP 371DTPA-NH—CH(2—O₂N-Phe-)-CH₂—CO-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1484

IMP 372 DTPA-Dpr(Ser)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1465

IMP 373 DTPA-Dpr(Sym-DTPA)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1753

IMP 374DTPA-Dpr(Cl—CH₂CO-Cys(Et)-)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1585

IMP 375DTPA-Dpr(2—Br-Phe-CHNH₂—CH₂—CO—)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1603 (FIG. 11)

IMP 376 DTPA-Cys(HO₃S—S)-D-Tyr-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1558

IMP 379 DTPA-Dpr(2—H₂N-Phe-CO-)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1497

IMP 382 DTPA-Dpr(H)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1378

IMP 383 DTPA-Dpr(Gla-)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1507

IMP 384DTPA-Dpr(2-HO-Phe-CHNH₂—CH₂—CO—)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1541 (FIG. 12)

IMP 385 DTPA-Dpr(Dpr)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1464

IMP 386DTPA-Dpr(2-pyridyl-CH₂—CHNH₂—CO—)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1526 (FIG. 13)

IMP 387DTPA-Dpr(D-9-anthrylalanine)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂MH⁺1625

IMP 389 DTPA-Dpr(2-carboxypiperizinyl)-D-Ala-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH₂ MH⁺1490 (FIG. 14)

IMP 460 NODA-GA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2 MH⁺1366

Further examples of peptides of possible use are shown in FIGS. 15 and16. FIG. 16 shows the structures of IMP 422, IMP 426 and IMP 428. Asdiscussed below, IMP 449 (FIG. 15) shows particular stability of theF-18 conjugated peptide under in vivo conditions, of use for labelingand imaging techniques.

FIG. 17 shows an alternative configuration for a NOTA type ligand. TheNOTA moiety could be made from D or L para-nitrophenylalanine and theiminodiacetic acid portion would come from diaminopropionic acid, whichcould be D or L. Furthermore, the position of the ethylene bridge couldbe switched with the diaminopropionic acid to give a differentconfiguration of groups on the ligand. All of these modifications couldaffect binding kinetics and stability of the complex, which issubsequently formed. FIG. 18 illustrates the structure of a NODA-Gapeptide that could be labeled with, for example, Ga-68 or F-18.

In certain embodiments, alternative chelating moieties may be used tobind to ¹⁸F-metal or ¹⁸F-boron complexes. FIG. 22A-D illustrates someexemplary potential chelating moieties based on the structure of NETA.As discussed above, Chong et al. (2007) report that NETA ligands mayshow improved serum stability when complexed with various metals.Chelator design may also be optimized to increase the binding affinityof the peptide for ¹⁸F-metal.

Results of Peptide Labeling Screening Study

Most of the DTPA derivatives showed labeling comparable to the labelingof IMP 272. There were exceptions, IMP 349, bearing the bisphosphonategroup on a cysteine side chain, labeled very poorly. The DOTA ligand didnot bind the Al-¹⁸F. The ITC DTPA ligand of IMP 326 did not bind theAl-¹⁸F as well as DTPA. The NTA ligand of IMP 331 did not bind theAl-¹⁸F. The EDTA ligand of IMP 332 bound the Al-¹⁸F but not as well asthe DTPA. Symmetrical DTPA ligand did not bind the Al-¹⁸F. Thephosphonates and phosphate groups tested did not bind Al-¹⁸F well underthe conditions tested. The screen did show that a group that wasattached near the DTPA could influence the stability of the Al-¹⁸F-DTPAcomplex. The screen showed that IMP 375 labeled better and formed acomplex that was significantly more stable than IMP 272. IMP 375 labeledwell and was stable in water, showing 95.4% remaining bound after 5hours at 25° C. (not shown). For in vivo use a peptide with high serumstability would be preferred.

The peptide labeling screening study only looked at the binding ofAl-¹⁸F. Some of the peptides that did not label well with Al-¹⁸F mightlabel better with another metal binding to the F-18.

Peptide Synthesis

The peptides were synthesized by solid phase peptide synthesis using theFmoc strategy. Groups were added to the side chains of diamino aminoacids by using Fmoc/Aloc protecting groups to allow differentialdeprotection. The Aloc groups were removed by the method of Dangles et.al. (J. Org. Chem. 1987, 52:4984-4993) except that piperidine was addedin a 1:1 ratio to the acetic acid used. The unsymmetrical tetra-t-butylDTPA was made as described in McBride et al. (US Patent Application Pub.No. US 2005/0002945 A1, application Ser. No. 10/776,470, Pub. Date. Jan.6, 2005). The tri-t-butyl DOTA, symmetrical tetra-t-butyl DTPA andITC-benzyl DTPA were obtained from MACROCYCLICS®. The Aloc/Fmoc Lysineand Dap (diaminopropionic acid derivatives (also Dpr)) were obtainedfrom CREOSALUS® or BACHEM®. The Sieber Amide resin was obtained fromNOVABIOCHEM®. The remaining Fmoc amino acids were obtained fromCREOSALUS®, BACHEM®, PEPTECH® or NOVABIOCHEM®.

IMP 272 was synthesized as described (McBride et al., US PatentApplication Publ. No. 20040241158 A1, application Ser. No. 10/768,707,Dec. 2, 2004). IMP 288 was made as described (McBride et al., J. Nucl.Med. 2006, 47:1678-1688).

IMP 326 The hydrazine peptide (IMP 319) was made on Sieber amide resinusing Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Glu(OBut)-OH, Fmoc-D-Lys(Aloc)-OH,Fmoc-D-Tyr(But)-OH and 4-(Boc-NH—NH—)C₆H₄-CO₂H in that order. The4-(Boc-NH—NH—)C₆H₄—CO₂H was made by adding Boc dicarbonate to4-hydrazinobenzoic acid in a dioxane sodium hydroxide solution.

After the addition of the Boc-hydrazide the side chain Aloc groups wereremoved and the Trityl-HSG-OH groups were added to the side chains ofthe lysines. The peptide was then cleaved from the resin with TFA andpurified by HPLC to obtain the desired hydrazine bis-HSG peptide IMP 319(MH⁺1201). The hydrazide peptide (0.0914 g) was then mixed with 0.0650 gof ITC-Benzyl DTPA in 3 mL of 0.1 M sodium phosphate pH 8.2. The pH ofthe solution was adjusted with 1 M NaOH to keep the pH at pH 8.2. Afterthe reaction between the peptide and the ITC-Benzyl DTPA was completethe peptide conjugate was purified by HPLC.

IMP 329 The deferoxamine isothiocyanate was prepared by mixing 1.0422 gof deferoxamine mesylate (1.59×10⁻³ mol) with 0.2835 g (1.59×10⁻³ mol)of thiocarbonyldiimidazole in 10 mL of 1:1 methanol/water.Triethylamine, 0.23 mL was added and the reaction was purified byreverse phase HPLC after 2.5 hr to obtain the deferoxamineisothiocyanate MNa+625.

The hydrazine peptide, IMP 319, (0.0533 g, 4.4×10⁻⁵ mol, MH⁺1201) wasmixed with 0.0291 g of deferoxamine isothiocyanate in a sodium phosphatebuffer at pH 8.1 for two hours then purified by HPLC to afford thedesired product MH+ 1804.

IMP 331 The following amino acids were attached to Sieber amide resin(0.58 mmol/g) in the order shown; Fmoc-D-Lys(Aloc)-OH,Fmoc-D-Tyr(But)-OH and Fmoc-D-Lys(Aloc)-OH. The Aloc groups were removedand Trt-HSG-OH was added to the side chains of the lysines. The Fmoc wasremoved, then Fmoc-D-Ala-OH and Fmoc-Asp-OBut were added in that order(0.5 g of resin). The Fmoc was removed and the nitrogen of the Asp wasalkylated overnight with 3 mL t-butyl bromoacetate and 3.6 mLdiisopropylethylamine in 3.4 mL of NMP. The peptide was cleaved from theresin with TFA and purified by reverse phase HPLC to obtain the desiredpeptide MH⁺1240.

IMP 332 The peptide was made on 3 g of Sieber amide resin (0.58 mmol/g).The following amino acids were added to the resin in the order shown:Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Tyr(But)-OH, Fmoc-D-Lys(Aloc)-OH,Fmoc-D-Ala-OH, and Fmoc-Dpr(Fmoc)-OH. The resin was split into portionsfor subsequent syntheses. One gram of the resin was removed and the Fmocgroups were removed from the diaminopropionic acid. The peptide wasalkylated overnight with 3 mL t-butyl bromoacetate, 3.6 mLdiisopropylethyl amine and 3.4 mL NMP. The side chain Aloc groups werethen removed and the Trt-HSG-OH groups were added. The peptide was thencleaved from the resin and purified by HPLC to obtain the productMH⁺1327.

IMP 333 The peptide was made with 1 g of the same resin that was used tomake IMP 332. The DTPA tetra-t-butyl ester (U.S. Publ. No. 20050002945)was added to both of the amines of the Dpr group. The Aloc groups werethen removed and the Trt-HSG-OH was added. The peptide was then cleavedand purified by HPLC to obtain the desired product MH⁺1845.

IMP 334 The peptide was made on 1 g Rink amide resin (0.7 mmol/g) withthe following amino acids added in the order shown: Fmoc-D-Lys(Aloc)-OH,Fmoc-D-Glu(But)-OH, Fmoc-D-Lys(Aloc)-OH, Boc-Ser(But)-OH, The Alocgroups were removed and the Trityl-HSG-OH was added. The peptide wascleaved from the resin with TFA. The crude peptide was collected byprecipitation from ether and dried. Sodium periodate, 0.33 g, wasdissolved in 15 mL water. The crude peptide was dissolved in 1 mL 0.5 Msodium phosphate pH 7.6, 3 mL water and 1 mL of the periodate solution.3 mL more periodate in one milliliter increments was added over ˜2 hr.The mixture was then purified by reverse phase HPLC and lyophilized toobtain the aldehyde IMP 289 HCO-CO-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂MH⁺959. Alendronate (0.0295 g, CALBIOCHEM® was dissolved in 150 μL 0.1 MNaOAc pH 4. The peptide, IMP 289, (0.0500 g) was dissolved in 100 μL of13% isopropanol in water. Sodium cyanoborohydride was added and themixture was purified by HPLC to afford the desired product MH⁺1192.

IMP 337 & IMP 338 The peptide was made on Sieber amide resin using thefollowing amino acids added in the order shown: Fmoc-D-Lys(Aloc)-OH,Fmoc-D-Ala-OH, Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Ala-OH,Fmoc-D-Ser(PO(OBzl)OH)-OH, Fmoc-D-Ser(PO(OBzl)OH)-OH, and Ac₂O. The Alocgroups were removed and the Trt-HSG-OH groups were added to the sidechains of the lysines. The peptide was cleaved from the resin andpurified by HPLC to afford the desired products: IMP 337 MH+1291 and IMP338 MH⁺1126.

IMP 345 The peptide was made on Sieber amide Resin using the followingamino acids added in the order shown: Fmoc-D-Lys(Aloc)-OH,Fmoc-D-Ala-OH, Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Ala-OH,Fmoc-D-Ser(PO(OBzl)OH)-OH, and tetra-t-butyl DTPA. The Aloc groups wereremoved and the Trt-HSG-OH groups were added to the side chains of thelysines. The peptide was cleaved from the resin and purified by HPLC toafford the desired product: IMP 345 MH⁺1459.

IMP 349 The peptide IMP 347DTPA-D-Cys-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ was made on Sieberamide Resin using the following amino acids added in the order shown:Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, Fmoc-D-Ala-OH,Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH were added, the Aloc was cleavedFmoc-D-Ala-OH, Fmoc-D-Cys(Trt)-OH and tetra-t-butyl DTPA were added. Thepeptide was cleaved from the resin and purified by HPLC to afford thedesired product: IMP 347 MH⁺1395. The peptide, IMP 347, 0.0446 g(3.2×10⁻5 mol) was mixed with 0.4605 g (2.4×10⁻³ mol) ofethenylidenebis(phosphonic acid) (Degenhardt et al., J. Org. Chem. 1986,51:3488-3490) in 3 mL of water and the solution was adjusted to pH 6.5with 1 M NaOH added dropwise. The reaction was stirred overnight and thereaction solution was adjusted to pH 1.49 by the addition of excessethenylidenebis(phosphonic acid). The mixture was stirred overnight atroom temperature and then purified by HPLC to obtain the desired peptideIMP 349 MH⁺1583.

IMP 361 The peptide was made on Sieber amide resin using the followingamino acids added in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH,the Aloc was cleaved, Fmoc-D-Ala-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OHwere added, the Aloc was cleaved, Fmoc-D-Ala-OH, Fmoc-Dap(Aloc)-OH andtetra-t-butyl DTPA were added. The Aloc on the side chain of the Dap wasremoved and bromo acetyl was added with bromo acetic anhydride. Thecrude product was purified by HPLC to obtain the desired peptide IMP 361(MH⁺1498).

IMP 366 The peptide was made by the same method as IMP 361 withphenylthioacetic acid added last. The crude product was purified by HPLCto afford the product IMP 366 MH⁺1528.

IMP 368 The peptide was as described for IMP 349 except the cysteineresidue was not added and symmetrical tetra-t-butylDTPA (MACROCYCLICS®)was used in place of the unsymmetrical DTPA to obtain the desiredproduct after purification, IMP 368 MH⁺1292.

IMP 369 The peptide was made as described for IMP 349 withFmoc-R-3-amino-3-(2-bromophenyl)propionic acid added in place of theD-Cys and symmetrical tetra-t-butylDTPA added in place of theunsymmetrical version to the DTPA tetra-t-butyl ester. The crude peptidewas purified to obtain the desired product, MH⁺1517.

IMP 370 The peptide was made as described for IMP 369 exceptFmoc-R-3-amino-3-(2-nitrophenyl) propionic acid was used instead of thebromo. The desired product was obtained after purification by HPLCMH⁺1484.

IMP 371 The peptide was made as described for IMP 370 except theunsymmetrical tetra-t-butyl DTPA was used in place of the of thesymmetrical version. The desired product was obtained after purificationby HPLC MH⁺1484.

IMP 372 The peptide was made as described for IMP 361 withFmoc-Ser(But)-OH used to attach the Ser to the Dap side chain. The Fmocwas removed and the peptide was cleaved from the resin and purified toobtain the desired product MH⁺1465.

IMP 373 The peptide was made as described for IMP 361 withsymmetrical-tetra-t-butylester DTPA used to attach the Sym-DTPA to theDap side chain. The peptide was cleaved from the resin and purified toobtain the desired product MH⁺1753.

IMP 374 The peptide was made as described for IMP 361 with Fmoc-S-ethylcysteine added to the Dap side chain followed by chloro acetyl (on thecysteine nitrogen) added via chloroacetic anhydride. The peptide wascleaved from the resin and purified to obtain the desired productMH⁺1585.

IMP 375 The peptide was made as described for IMP 361 withFmoc-R-3-amino-3-(2-bromophenyl)propionic acid added to the Dap sidechain followed by cleavage of the Fmoc group. The peptide was cleavedfrom the resin and purified to obtain the desired product MH⁺1603.

IMP 376 The peptide was made as described for IMP 361 withFmoc-D-Tyr(But)-OH added after the second alanine followed byFmoc-Cys(SO₃H) and tetra-t-butylDTPA. The peptide was cleaved from theresin and purified to obtain the desired product MH⁺1558.

IMP 379 The peptide was made as described for IMP 361 with Boc-2-Abz-OHadded to the side chain of the Dap. The peptide was cleaved from theresin and purified to obtain the desired product MH⁺1497.

IMP 382 The peptide was made as described for IMP 361 with the Alocremoved from the side chain of the Dap. The peptide was cleaved from theresin and purified to obtain the desired product MH⁺1378.

IMP 383 The peptide was made as described for IMP 361 withFmoc-Gla(OBut)₂-OH added to the side chain of the Dap. The peptide wascleaved from the resin and purified to obtain the desired productMH⁺—CO₂ 1507

IMP 384 The peptide was made as described for IMP 361 withFmoc-Boc-S-3-amino-3-(2-hydroxyphenyl)propionic acid added to the sidechain of the Dap. The peptide was cleaved from the resin and purified toobtain the desired product MH⁺1541.

IMP 385 The peptide was made as described for IMP 361 withFmoc-Dpr(Fmoc)-OH added to the side chain of the Dap. The peptide wascleaved from the resin and purified to obtain the desired productMH⁺1464.

IMP 386 The peptide was made as described for IMP 361 withBoc-D-2-pyridylalanine-OH added to the side chain of the Dap. Thepeptide was cleaved from the resin and purified to obtain the desiredproduct MH⁺1526.

IMP 387 The peptide was made as described for IMP 361 withFmoc-D-9-anthrylalanine-OH added to the side chain of the Dap. Thepeptide was cleaved from the resin and purified to obtain the desiredproduct MH⁺1625.

IMP 389 The peptide was made as described for IMP 361 withbis-Boc-piperazine-2-carboxylate added to the side chain of the Dap. Thepeptide was cleaved from the resin and purified to obtain the desiredproduct MH⁺1664.

Example 5 Alternative Methods for Preparing and Separating F-18 LabeledPeptides

In certain embodiments, heating is used to get the Al-F-18 complex intothe NOTA chelating group. Alternatively, ITC benzyl NOTA (Macrocyclics)could be labeled with Al-F-18 and then conjugated to other heatsensitive molecules, such as proteins, after labeling. If high specificactivity is needed the ITC Benzyl NOTA complex can be purified away fromthe cold ligand.

Al was added to the peptide and its HPLC profile compared to the emptyNOTA peptide and the Al-F-18 peptide. The Al peptide and the Al-F-18peptides have virtually the same retention time by HPLC, with ˜1 minlonger RT for the unlabeled peptide. The peptide was purified on aPHENOMENEX™ ONYX® monolithic C-18 100×4.5 mm column using a 3 mL/minflow rate. Buffer A was 0.1% TFA in water and Buffer B was 90% CH₃CN 10%water and 0.1% TFA. The linear gradient went from 100% buffer A to 75:25A/B over 15 min. Since the Al complex co-elutes with the Al-F-18complex, the amount of Al and F-18 added will determine the specificactivity.

IMP 449 was prepared according to Example 7 below and labeled asfollows. The F-18 was received in a 2.0 mL Fisher Microcentrifuge vial(02-681-374) containing 15 mCi of F-18 in ˜325 μL in water. 3 ˜L of 2 mMAlCl₃ in 0.1 M pH 4 NaOAc was added to the F-18 solution and then vortexmixed. After about 4 min, 10 μL of 0.05 M IMP 449 in pH4 0.5 M NaOAc wasadded. The sample was vortex mixed again and heated in a 102° C. heatingblock for 17 min. The reaction was then cooled briefly and then the vialcontents were removed and purified by HPLC as described above.

Separately, elution conditions were determined on the WATERS(® ALLIANCE™analytical system and the labeled peptide was eluted between 7.5 and 8.5min. The analytical HPLC showed that the labeled peptide contained theAl-F IMP 449 (UV 220 nm) and did not contain the uncomplexed peptide,resulting in an increased specific activity.

The peptide was diluted in water and then pushed through a WATERS® OASISPLUS HLB™ extraction column. The labeled peptide was eluted with 3 mL of1:1 EtOH/H₂O. HPLC analysis of the eluents confirmed that the columnefficiently trapped the labeled peptide, which allowed the acetonitrileand TFA to be washed away from the peptide. The HPLC also showed that1:1 EtOH/H₂O eluent contained the desired product free of loose F-18 ina solvent suitable for injection after dilution. The apparent yieldafter purification was 11%.

Example 6 In-Vivo Studies

Nude mice bearing GW-39 human colonic xenograft tumors (100-500 mg) areinjected with the bispecific antibody hMN-14 x m679 (1.5×10⁻¹⁰ mol). Theantibody is allowed to clear for 24 hr before the F-18 labeledHSG-bearing peptide (8.8 μCi, 1.5×10⁻¹¹ mol) is injected. The animalsare imaged at 3, 24 and 48 hr post injection. The xenograft tumors areclearly imaged by PET scanning detection of the F-18 labeled peptidebound to the bispecific hMN-14 x m679 that is localized to the tumors bybinding of hMN-14 to tumor antigen.

Example 7 Production and Use of a Serum-Stable F-18 Labeled Peptide

IMP 449 NOTA-ITC benzyl-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺1459(FIG. 15)

The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺1009 wasmade on Sieber Amide resin by adding the following amino acids to theresin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc wascleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make the desiredpeptide. The peptide was then cleaved from the resin and purified byHPLC to produce IMP 448, which was then coupled to ITC-benzyl NOTA. Thepeptide, IMP 448, 0.0757g (7.5×10⁻⁵ mol) was mixed with 0.0509 g(9.09×10⁻⁵ mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassiumcarbonate anhydrous (0.2171 g) was then slowly added to the stirredpeptide/NOTA solution. The reaction solution was pH 10.6 after theaddition of all the carbonate. The reaction was allowed to stir at roomtemperature overnight. The reaction was carefully quenched with 1 M HClafter 14 hr and purified by HPLC to obtain 48 mg of IMP 449, the desiredproduct (FIG. 15).

F-18 Labeling of IMP 449

The peptide IMP 449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2mM peptide solution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mMsolution of Al in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi ofF-18. The solution was then mixed with 20 μL of the 2 mM IMP 449solution and heated at 105° C. for 15 min. Reverse Phase HPLC analysisshowed 35% (RT ˜10 min) of the activity was attached to the peptide and65% of the activity was eluted at the void volume of the column (3.1min, not shown) indicating that the majority of activity was notassociated with the peptide. The crude labeled mixture (5 μL) was mixedwith pooled human serum and incubated at 37° C. An aliquot was removedafter 15 min and analyzed by HPLC. The HPLC showed 9.8% of the activitywas still attached to the peptide (down from 35%). Another aliquot wasremoved after 1 hr and analyzed by HPLC. The HPLC showed 7.6% of theactivity was still attached to the peptide (down from 35%), which wasessentially the same as the 15 min trace (data not shown).

High Dose F-18 Labeling

Further studies with purified IMP 449 demonstrated that the F-18 labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. These results demonstrate thatthe F-18 labeled peptides disclosed herein exhibit sufficient stabilityunder approximated in vivo conditions to be used for F-18 imagingstudies.

F-18 ˜21 mCi in ˜400 μL of water was mixed with 9 μL of 2 mM AlCl₃ in0.1 M pH 4 NaOAc. The peptide, IMP 449, 60 μL (0.01 M, 6×10⁻⁷ mol in 0.5NaOH pH 4.13) was added and the solution was heated to 110° C. for 15min. The crude labeled peptide was then purified by placing the reactionsolution in the barrel of a 1 cc WATERS® HLB column and eluting withwater to remove unbound F-18 followed by 1:1 EtOH/H2O to elute the F-18labeled peptide. The crude reaction solution was pulled through thecolumn into a waste vial and the column was washed with three onemilliliter fractions of water (18.97 mCi). The HLB column was thenplaced on a new vial and eluted with two×200 μL 1:1 EtOH/H₂O to collectthe labeled peptide (1.83 mCi). The column retained 0.1 mCi of activityafter all of the elutions were complete. An aliquot of the purified F-18labeled peptide (20 μL) was mixed with 200 μL of pooled human serum andheated at 37° C. Aliquots were analyzed by reverse phase HPLC (asdescribed above). The results showed the relative stability of F-18labeled purified IMP 449 at 37° C. at time zero, one hour (91% labeledpeptide), two hours (77% labeled peptide) and four hours (76% labeledpeptide) of incubation in human serum (not shown). It was also observedthat F-18 labeled IMP 449 was stable in TFA solution, which isoccasionally used during reverse phase HPLC chromatography. Thereappears to be a general correlation between stability in TFA andstability in human serum observed for the exemplary F-18 labeledmolecules described herein. These results demonstrate that F-18 labeledpeptide, produced according to the methods disclosed herein, showssufficient stability in human serum to be successfully used for in vivolabeling and imaging studies, for example using PET scanning to detectlabeled cells or tissues.

Example 8 In Vivo Biodistribution of F-18 Labeled IMP 449 in SCID Mice

F-18 labeled IMP 449 was prepared as described above (Example 7). Thematerial was purified on an OASIS® HLB column (WATERS®, Milford, Mass.).The unbound material was washed out with water and the labeled peptidethat was bound to the column was eluted with 1:1 ethanol:water mixture.Both fractions were analyzed by reverse phase C18 HPLC. The purifiedpeptide eluted as several peaks on the reverse HPLC column (not shown).The unbound fraction collected from the OASIS® column showed poorrecovery, 7%, from the C18 column (not shown).

The “unbound” fraction and the purified ¹⁸F-IMP 449 were injected intoSCID mice that were previously injected with sc SU-DHL6 lymphoma cells.Only a few of the mice had visible tumors. Biodistribution data showed asignificant difference between the “unbound” F-18 fraction and thepurified ¹⁸F-IMP 449. Data are shown in Tables 4-6 below. Note that inthis study, no pretargeting bispecific antibodies were administered tothe animals before the labeled peptide. These results demonstrate thedistribution of labeled peptide vs. free F-18 in vivo.

Unconjugated F-18 shows a high level of distribution to bone tissue invivo. Uptake 20 minutes after injection was, as expected, seen primarilyin the bone (spine), with about 12-15% injected dose per gram (ID/g),followed by the kidneys with about 4% ID/g. Localization of the F-18label to bone tissue was substantially decreased by conjugation to atargeting peptide. When bound to IMP 449, uptake in the bone is reducedto ˜1% ID/g at 20 min and 0.3% at 1 h after injection, with renal uptakeof 11% at 20 min and 3.3% ID/g at 1 hr. Renal uptake of the peptidealone was similar to that of the pretargeted ¹⁸F-IMP 449 peptide (seefollowing Example), suggesting its uptake was a function of the peptiderather than a consequence of the animals having been give the bsMAb 18 hearlier. Relatively low non-specific uptake was observed in the spineand femur with the F-18 labeled peptide compared with unbound F-18.

TABLE 4 F-18 “unbound” fraction at 20 min post injection: % ID/g meanand the individual animals. Tissue n Mean SD Animal 1 Animal 2 Animal 3Tumor 1 — — 0.902 — — Liver 3 2.056 0.244 1.895 2.338 1.937 Spleen 31.869 0.434 1.677 2.366 1.564 Kidney 3 4.326 0.536 3.931 4.936 4.111Lung 3 2.021 0.149 1.903 2.188 1.972 Blood 3 2.421 0.248 2.355 2.6962.212 Stomach 3 0.777 0.409 0.421 1.224 0.687 Small Int. 3 2.185 0.1422.042 2.325 2.187 Large Int. 3 1.403 0.069 1.482 1.356 1.372 Femur 311.688 1.519 11.502 13.292 10.270 Spine 3 14.343 2.757 17.506 13.07212.452 Muscle 3 1.375 0.160 1.191 1.457 1.478

TABLE 5 ¹⁸F-IMP 449 purified, 80 μCi, 1 × 10⁻⁸ mol at 20 min postinjection: % ID/g mean and the individual animals Tissue n Mean SDAnimal 1 Animal 2 Animal 3 Animal 4 Animal 5 Tumor 1 — — 0.891 — — — —Liver 5 2.050 0.312 1.672 1.801 2.211 2.129 2.440 Spleen 5 1.297 0.2590.948 1.348 1.144 1.621 1.425 Kidney 5 12.120 4.128 8.354 7.518 12.49215.535 16.702 Lung 5 2.580 0.518 2.034 2.103 2.804 2.678 3.278 Blood 53.230 0.638 2.608 2.524 3.516 3.512 3.992 Stomach 5 1.017 0.907 0.8050.775 0.344 0.557 2.605 Small Int. 5 1.212 0.636 0.896 0.921 0.927 0.9672.349 Large Int. 5 0.709 0.220 0.526 0.568 0.599 0.793 1.057 Femur 50.804 0.389 0.314 0.560 1.280 0.776 1.087 Spine 5 3.915 6.384 0.8190.923 1.325 1.177 15.330^(#) Muscle 5 0.668 0.226 0.457 0.439 0.9600.673 0.814 ^(#)High spine uptake in Animal #5 was confirmed byrecounting.

TABLE 6 ¹⁸F-IMP 449 purified, 80 μCi, 1 × 10⁻⁸ mol at 1 h postinjection: % ID/g mean and the individual animals Ani- Ani- Tissue nMean SD Animal 1 Animal 2 mal 3 mal 4 Tumor 1 0.032 0.064 0.000 0.1270.000 0.000 Liver 4 0.883 0.308 1.103 0.632 0.604 1.191 Spleen 4 1.0610.702 1.598 0.631 0.301 1.713 Kidney 4 3.256 0.591 3.606 2.392 3.3623.666 Lung 4 0.324 0.094 0.411 0.232 0.256 0.399 Blood 4 0.285 0.1040.378 0.153 0.250 0.358 Stomach 4 0.152 0.082 0.225 0.041 0.199 0.142Small Int. 4 1.290 0.228 1.124 1.247 1.166 1.624 Large Int. 4 0.1150.035 0.167 0.091 0.094 0.109 Femur 4 1.006 0.876 2.266 0.448 0.9390.374 Spine 4 0.314 0.076 0.423 0.257 0.268 0.306 Muscle 4 0.591 0.9460.205 0.077 2.008 0.075

We conclude that the F-18 labeled peptide showed sufficient in vivostability to successfully perform labeling and imaging studies.

Example 9 In vivo Studies With Pretargeting Antibody

F-18 labeled IMP 449 was prepared as follows. The F-18, 54.7 mCi in ˜0.5mL was mixed with 3 μL 2 mM Al in 0.1 M NaOAc pH 4 buffer. After 3min,10 μL of 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer was added and thereaction was heated in a 96° C. heating block for 15 min. The contentsof the reaction were removed with a syringe. The crude labeled peptidewas then purified by HPLC on a Phenomenex Onyx monolithic C18, 100×4.6mm column part. No. CHO-7643. The flow rate was 3 mL/min. Buffer A was0.1% TFA in water and Buffer B was 90 % acetonitrile in water with 0.1%TFA. The gradient went from 100% A to 75/25 A:B over 15 min. There wasabout 1 min difference in RT between the labeled peptide, which elutedfirst and the unlabeled peptide. The HPLC eluent was collected in 0.5min fractions. The labeled peptide came out between 6 to 9 min dependingon the HPLC used. The HPLC purified peptide sample was further processedby diluting the fractions of interest two fold in water and placing thesolution in the barrel of a 1 cc Waters HLB column. The cartridge waseluted with 3×1 mL water to remove acetonitrile and TFA followed by 400μL 1:1 EtOH/H2O to elute the F-18 labeled peptide.

The purified ¹⁸F-IMP 449 eluted as a single peak on an analytical HPLCC18 column.

Taconic nude mice bearing the four slow-growing sc CaPan1 xenograftswere used. Three of the mice were injected with TF10 (162 μg) followedwith ¹⁸F-IMP 449 18 h later. TF10 is a humanized bispecific antibody ofuse for tumor imaging studies, with divalent binding to the PAM-4defined MUC 1 tumor antigen and monovalent binding to HSG (see, e.g.,Gold et al., 2007, J. Clin. Oncol. 25(18S):4564). One mouse was injectedwith peptide alone. All of the mice were necropsied 1 h post peptideinjection. Tissues were counted immediately. Animal #2 showed highcounts in the femur. The femur was transferred into a new vial and wasrecounted along with the old empty vial. Recounting indicated that thecounts were on the tissue. This femur was broken and had a large pieceof muscle attached to it. Comparison of mean distributions showedsubstantially higher levels of F-18-labeled peptide localized in thetumor than in any normal tissues in the presence of tumor-targetingbispecific antibody.

Tissue uptake was similar in animals given the ¹⁸F-IMP 449 alone or in apretargeting setting. Uptake in the human pancreatic cancer xenograft,CaPan1, at 1 h was increased 5-fold in the pretargeted animals ascompared to the peptide alone (4.6±0.9% ID/g vs. 0.89% ID/g).Exceptional tumor/nontumor ratios were achieved at this time (e.g.,tumor/blood and liver ratios were 23.4±2.0 and 23.5±2.8, respectively).

TABLE 7 Tissue uptake at 1 h post peptide injection, mean and theindividual animals: TF10 (162 μg) -→18 h → ¹⁸F IMP449 ¹⁸F IMP (10:1) 449alone Tissue n Mean SD Animal 1 Animal 2 Animal 3 Animal 1 Tumor 3 4.5910.854 4.330 5.546 3.898 0.893 (mass) (0.675 g) (0.306 g) (0.353 g)(0.721 g) Liver 3 0.197 0.041 0.163 0.242 0.186 0.253 Spleen 3 0.2020.022 0.180 0.224 0.200 0.226 Kidney 3 5.624 0.531 5.513 6.202 5.1585.744 Lung 3 0.421 0.197 0.352 0.643 0.268 0.474 Blood 3 0.196 0.0280.204 0.219 0.165 0.360 Stomach 3 0.123 0.046 0.080 0.172 0.118 0.329Small Int. 3 0.248 0.042 0.218 0.295 0.230 0.392 Large 3 0.141 0.0940.065 0.247 0.112 0.113 Int. Pancreas 3 0.185 0.078 0.259 0.194 0.1030.174 Spine 3 0.394 0.427 0.140 0.888 0.155 0.239 Femur 3 3.899 4.0982.577 8.494* 0.625 0.237 Brain 3 0.064 0.041 0.020 0.072 0.100 0.075Muscle 3 0.696 0.761 0.077 1.545 0.465 0.162 *High counts in Animal # 2femur were confirmed by recounting after transferring femur into a newvial. Animal #2 showed higher uptake in normal tissues than Animals #1and #3.

Example 10 Comparison of Biodistribution of ¹¹¹In-IMP 449 vs. ¹⁸F-IMP449 With Pretargeting Antibody

The goal of the study was to compare biodistribution of ¹¹¹In-IMP 449and ¹⁸F-IMP 449 in nude mice bearing sc LS 174 T xenografts afterpretargeting with bispecific antibody TF2. TF2 antibody was made by thedock-and-lock method and contains binding sites for the CEA tumorantigen and the HSG hapten (see, e.g., Sharkey et al., Radiology 2008,246:497-507; Rossi et al., PNAS USA 2006, 103:6841-46). Since there wereinsufficient numbers of mice with tumors at one time, the study wasperformed on 2 different weeks.

¹¹¹In-IMP 449: ¹¹¹In labeling was performed using a procedure similar tothe one used for labeling IMP 288, except at lower specific activity.ITLC and C-18 RP HPLC showed ˜30% unbound (not shown). The labeledpeptide was purified on an HLB column (1 mL, 30 mg). The analyses of thepurified product again showed 33% unbound (top 20% of strip) by ITLCdeveloped in saturated sodium chloride. RP HPLC showed multiple peaksbefore and after purification (not shown). SE HPLC after purificationshowed 47% of the activity shift to HMW when mixed with 20× molar excessof TF2 (not shown).

¹⁸F-IMP 449: Labeling was performed as described above except the F-18was purified on a QMA cartridge before labeling as described by others(Kim et. al. Applied Radiation and Isotopes 61, 2004, 1241-46). Briefly,the Sep-Pak® Light Waters Accell™ Plus QMA Cartridge was preparedflushed with 10 mL 0.4 M KHCO₃ and then washed with 10 mL DI water. The¹⁸F (42 mCi) in 2 mL water was loaded onto the QMA cartridge. Thecartridge was eluted with 10 mL DI water to remove impurities. Thecolumn was then eluted with 1 mL 0.4 M KHCO₃ in 200 μL fractions.Fraction number two contained the bulk of the activity, 33 mCi. The pHof the F-18 solution was then adjusted with 10 μL of glacial aceticacid. The ¹⁸F from fraction #2 was then mixed with 3 μL of 2 mM Al in0.1 M pH 4 NaOAc buffer. The sample was then mixed with 10 μL of 0.05 MIMP 449 in 0.5 M NaOAc buffer at pH4 and the reaction solution washeated at 94° C. for 15 min. The ¹⁸F-IMP 449 was purified by RP HPLC.The fraction containing the product was put through an HLB column toexchange the buffer. The column was washed with water after loading thesample. The product was eluted with 1:1 water:ethanol in a 400 μLvolume. RP HPLC of the product showed one major peak with a shoulder(not shown). Since the yield was low, the specific activity was low andmore peptide was injected into mice, resulting in a bsMAb:peptide ratioof 6.9:1 instead of 10:1.

Results

The labeling of IMP 449 with In-111 resulted in multiple products.Possibly some might be binuclear complexes. The ¹¹¹In-IMP 449 showedhigh kidney uptake and high blood concentration. However, even asmultiple species, ¹¹¹In-IMP 449 showed localization to the tumor whenpretargeted with TF2 (FIG. 19).

FIG. 19 shows the comparative biodistribution of In-111 and F-18 labeledIMP 449 in mice. Both labeled peptides showed similarly high levels oflocalization to tumor tissues in the presence of the bispecific TF2antibody. The In-I111 labeled species showed higher concentration inkidney than the F-18 labeled species in the presence or absence of TF2antibody. The data are summarized in Tables 8-11 below.

TABLE 8 Mice were injected with TF2 (163.2ug, 1.035 × 10⁻⁹ mol) ivfollowed with ¹¹¹In IMP 449 (1.035 × 10⁻¹⁰ mol) 16 h later. Peptidetissue uptake (% ID/g) at 1 h post peptide injection is shown below.Tissue n Mean SD Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 Tumor 59.18 1.02 9.22 8.47 8.04 9.45 10.70 Liver 5 1.15 0.09 1.03 1.25 1.201.21 1.08 Spleen 5 0.48 0.06 0.43 0.49 0.58 0.50 0.42 Kidney 5 6.63 1.388.81 6.21 7.03 5.85 5.23 Lung 5 1.03 0.14 0.92 1.14 1.18 1.04 0.86 Blood5 0.99 0.15 1.04 1.13 1.12 0.83 0.83 Stomach 5 0.16 0.05 0.25 0.17 0.160.13 0.12 Small Int. 5 2.33 0.65 2.21 2.51 2.01 3.33 1.59 Large Int. 50.20 0.04 0.21 0.25 0.18 0.21 0.14 Femur 5 1.45 0.87 0.59 1.30 0.71 2.022.62 Spine 5 1.18 1.23 0.89 3.35 0.76 0.47 0.43 Brain 5 0.14 0.16 0.050.06 0.13 0.04 0.43 Muscle 5 0.83 0.66 0.25 1.30 0.23 0.65 1.73 Body Wt.5 25.49 1.41 27.89 24.14 25.27 25.10 25.06

TABLE 9 A group of 2 mice were injected with ¹¹¹In IMP 449 (1.035 ×10⁻¹⁰ mol) without pretargeting antibody. Peptide tissue uptake (% ID/g)at 1 h post peptide injection is shown below. Tissue n Mean SD Animal 1Animal 2 Tumor 2 0.922 0.195 0.784 1.060 Liver 2 1.033 0.048 0.999 1.067Spleen 2 0.409 0.067 0.362 0.456 Kidney 2 6.046 0.449 5.729 6.364 Lung 20.695 0.032 0.672 0.717 Blood 2 0.805 0.182 0.934 0.676 Stomach 2 0.2900.055 0.251 0.329 Small Int. 2 2.234 0.594 1.814 2.654 Large Int. 20.237 0.022 0.253 0.222 Femur 2 1.210 1.072 1.968 0.453 Spine 2 1.4631.213 2.320 0.605 Brain 2 0.133 0.091 0.068 0.197 Muscle 2 1.005 1.1481.817 0.193 Body Wt. 2 26.65 3.19 28.90 24.39

TABLE 10 Mice were injected with TF2 (163.2ug, 1.035 × 10⁻⁹ mol) ivfollowed with ¹⁸F-IMP 449 (1.5 × 10⁻¹⁰ mol) 16 h later. Peptide tissueuptake (% ID/g) at 1 h post peptide injection is shown below. Tissue nMean SD Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 Tumor 5 7.624 3.0805.298 7.848 12.719 5.118 7.136 Liver 5 0.172 0.033 0.208 0.143 0.1960.131 0.180 Spleen 5 0.142 0.059 0.239 0.081 0.132 0.118 0.140 Kidney 52.191 0.125 2.313 2.141 2.154 2.319 2.027 Lung 5 0.315 0.094 0.474 0.2300.300 0.305 0.265 Blood 5 0.269 0.143 0.431 0.395 0.132 0.126 0.260Stomach 5 0.218 0.341 0.827 0.041 0.098 0.054 0.070 Small Int. 5 0.3510.313 0.903 0.185 0.297 0.170 0.198 Large Int. 5 0.069 0.028 0.076 0.0430.111 0.073 0.042 Femur 5 0.625 0.358 0.869 0.146 0.811 0.957 0.344Spine 5 0.585 0.569 0.159 0.119 0.493 1.526 0.626 Brain 5 0.029 0.0050.033 0.021 0.035 0.026 0.028 Muscle 5 0.736 0.970 0.190 0.064 0.4942.438 0.496 Body Wt. 5 24.69 1.20 23.05 26.36 24.45 24.48 25.11

TABLE 11 Mice were injected with ¹⁸F-IMP 449 (1.5 × 10⁻¹⁰ mol) withoutpretargeting antibody. Peptide tissue uptake (% ID/g) at 1 h postpeptide injection is shown below. Tissue n Mean SD Animal 1 Animal 2Animal 3 Animal 4 Animal 5 Tumor 5 0.472 0.201 0.256 0.344 0.533 0.4470.779 Liver 5 0.177 0.035 0.141 0.200 0.141 0.185 0.217 Spleen 5 0.1180.027 0.098 0.094 0.101 0.144 0.151 Kidney 5 2.727 0.367 2.430 2.4522.500 3.080 3.173 Lung 5 0.246 0.082 0.206 0.209 0.156 0.301 0.358 Blood5 0.167 0.072 0.110 0.135 0.104 0.217 0.267 Stomach 5 0.114 0.083 0.1490.241 0.037 0.067 0.074 Small Int. 5 0.277 0.081 0.407 0.286 0.206 0.2130.271 Large Int. 5 0.072 0.029 0.061 0.052 0.047 0.083 0.118 Femur 50.100 0.032 0.080 0.144 0.110 0.109 0.059 Spine 5 0.305 0.268 0.1040.647 0.099 0.132 0.545 Brain 5 0.034 0.025 0.018 0.018 0.022 0.0340.077 Muscle 5 0.088 0.022 0.087 0.069 0.069 0.122 0.092 Body Wt. 525.34 1.72 25.05 26.88 26.40 25.88 22.51

In summary, a simple, reproducible method and compositions are describedherein for producing F-18 labeled targeting peptides that are suitablefor use in in vivo imaging of a variety of disease states. The skilledartisan will realize that the bispecific antibodies disclosed above arenot limiting, but may comprise any known antibodies against a widevariety of disease or pathogen target antigens. Nor is the methodlimited to pretargeting with bispecific antibodies. In otherembodiments, molecules or complexes that directly bind to target cells,tissues or organisms to be imaged may be labeled with F-18 using themethods disclosed herein and administered to a subject for PET imaging(see Examples below).

The Al-F-18 labeled peptides, exemplified by IMP 449, are sufficientlystable under in vivo conditions to be utilized in known imagingprotocols, such as PET scanning. The present yield of radiolabeledpeptide prepared as described above varies between 5 and 20%, and evenwith a brief HPLC purification step to separate labeled from unlabeledpeptide the final yield is about 5%. Further, the claimed methods resultin preparation of F-18 labeled targeting peptides that are ready forinjection within 1 hour of preparation time, well within the decay timeof F-18 to allow suitable imaging procedures to be performed. Finally,the described and claimed methods result in minimal exposure of theoperator to radioisotope exposure, compared with known methods ofpreparing F-18 labeled compounds for imaging studies.

Example 11 F-18 Labeling Kit

An F-18 labeling kit was made by mixing 8.0 mg of IMP 449 with 0.1549 gof ascorbic acid. The two reagents were dissolved in 10.5 mL water andthe solution was dispensed in 1.0 mL aliquots into 10 vials. The pH wasnot adjusted. The solutions were frozen, lyophilized and sealed undervacuum.

Example 12 Imaging of Tumors In Vivo Using Labeled Peptides andPretargeting with Bispecific Antibodies

The present Examples show that in vivo imaging techniques usingpretargeting with bispecific antibodies and labeled targeting peptidesmay be used to successfully detect tumors of relatively small size. Thepretargeting antibodies utilized were either TF2, described above, orthe TF10 antibody.

Formulation Buffer:

The formulation buffer was made by mixing 0.3023 g ascorbic acid, 18.4mL DI water and 1.6 mL 1 M NaOH to adjust the pH to pH 6.61. The bufferwas dispensed in 1 mL aliquots into 20 vials and lyophilized.

The F-18 was purified on a WATERS® ACCELL™ Plus QMA Light cartridgeaccording to the literature procedure, wherein the cartridge was washedwith 10 mL 0.4 M KHCO₃ followed by a 10 mL wash with DI water. The F-18in 2 mL of water was pushed through the cartridge and then washed with10 mL of water. The F-18 was then eluted from the cartridge in 5×200 μLaliquots with 0.4 M KHCO₃. Most of the activity was eluted in the secondfraction. The activity in the second fraction was mixed with 3 μL 2 mMAl in a pH 4 acetate buffer. The Al-F-18 solution was then injected intothe ascorbic acid IMP 449 labeling vial and heated to 105° C. for 15min. The reaction solution was cooled and mixed with 0.8 mL DI water.The reaction contents were placed on a WATERS® OASIS® 1 cc HLB Columnand eluted into a waste vial. The column was washed with 3×1 mL DIwater. The column was transferred to a formulation vial containingascorbic acid. The column was eluted with 2×200 μL 1:1 EtOH/H₂O to elutethe labeled peptide.

Production of TF10 Bispecific Antibody Using DNL Technology

The cancer-targeting antibody component in TF10 is derived from hPAM4, ahumanized anti-MUC 1 MAb that has been studied as a radiolabeled MAb indetail (e.g. Gold et al., Clin. Cancer Res. 13: 7380-7387, 2007). Thehapten-binding component is derived from h679, a humanizedanti-histaminyl-succinyl-glycine (HSG) MAb discussed above. The TF10bispecific ([hPAM4]₂ x h679) antibody was produced using the methoddisclosed for production of the (anti CEA)₂ x anti HSG bsAb TF2 (Rossiet al., 2006). The TF10 construct bears two humanized PAM4 Fabs and onehumanized 679 Fab.

For TF10, a Fab of the humanized hPAM4 antibody was linked using apeptide spacer to an α-sequence. The α-sequence is unique because itspontaneously associates with another α-sequence to form a dimer. InTF10, the structure contains 2 hPAM4 anti-MUC1 Fabs linked together bythe 2 α-sequences (called hPAM4-DDD). The other component of TF10 isproduced by linking a β-sequence to the Fab′ of the humanized anti-HSGantibody. Unlike the α-sequence, the β-sequence does not self-associate,but instead binds to the dimeric structure formed by the 2 α-sequences(h679-AD). Thus, when these 2 separately produced proteins are mixedtogether, they immediately form an ‘a₂b’ structure, with each Fab′oriented in a manner to allow unimpeded binding to its antigen. Thestability of this binding interaction has been further improved bystrategically positioning cysteine in each of the α- and β-sequences (2in the β-sequence and 1 in the α-sequence). Because ‘b’ binds to ‘a₂’ ina highly specific orientation, once a₂b is assembled, disulfide bridgescan form between the α- and β-moieties, thereby covalently attachingthese 2 proteins. Both the α- and β-sequences are found in humanproteins, and therefore are not expected to add to the immunogenicity ofthe complex.

The anti-MUC1 fusion protein hPAM4-α was generated by fusion of the αsequence to the C-terminal end of the Fd chain. The anti-HSG fusionprotein h679-β was formed by linking the β sequence to the C-terminalend of the Fd chain. The stably tethered, multivalent bsMAb TF10 wasformed by pairing the hPAM4-α with the h679-β.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-α. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

A full tissue histology and blood cell binding panel has already beenexamined for hPAM4 IgG and for an anti-CEA x anti-HSG bsMAb that isentering clinical trials. hPAM4 binding was restricted to very weakbinding to the urinary bladder and stomach in ⅓ specimens (no bindingwas seen in vivo), and no binding to normal tissues was attributed tothe anti-CEA×anti-HSG bsMAb. Furthermore, in vitro studies against celllines bearing the H1 and H2 histamine receptors showed no antagonisticor agonistic activity with the IMP-288 di-HSG peptide, and animalstudies in 2 different species showed no pharmacologic activity of thepeptide related to the histamine component at doses 20,000 times higherthan that used for imaging. Thus, the HSG-histamine derivative does nothave pharmacologic activity.

Biodistribution, Targeting and Dosage Studies of TF10 BispecificAntibody

The biodistribution and tumor targeting of TF10 with increasing TF10doses is determined. These studies provide basic Pk data for TF10 over arange of doses. The primary dose range simulates human equivalent doses(HED) between 1.0 to 50 mg given to a 70 kg patient. Based on FDAguidelines for converting a dose given to an animal to a HED [i.e.,(mg/kg in a mouse/12.3)=mg/kg HED], a 1 mg (6.37 nmol) TF10 dose givento a 70 kg human would be equivalent to a 3.5 μg (0.022 nmol) dose in a20 g mouse.

Briefly, animals are given iv injections of 3.5, 17.5, 35, and 70 TF10(trace ¹²⁵I-TF10 added). Animals given 17.5, 35, and 70 μg doses (HED=1,5, 10 and 20 mg) are necropsied at 1, 6, 16, 48, and 72 h (n=5 perobservation; total N=75 animals/cell line). Studies with the current lotof TF10 have indicated a very rapid clearance in mice, similar to thatof the TF2 anti-CEA construct described above.

Pk studies are also performed with ¹³¹I-TF10 in rabbits. Prior studieswith TF2 anti-CEA bsMAb have indicated that rabbits might better predictthe Pk behavior that is observed in patients, since they clear humanizedanti-CEA IgG in an identical manner as that found in patients, whilemice clear humanized IgG at a faster rate. These studies would involve 4rabbits, 2 given a 5-mg HED and 2 given a 20-mg HED of TF 10 spiked with¹³¹I-TF10 (˜700 μCi). Rabbits are bled at 5 min, 1, 3, 6, 24, 48, 72,96, 120, and 168 h. Whole-body images are also taken using an ADAC Solusgamma camera equipped with a high-energy collimator. An ¹³¹I-standard(˜20 μCi in a 10 mL syringe) is placed in the field of view with eachrabbit during each imaging session taken at 3, 24, 48, 120, and 168 h.The standard is then used to provide semi-quantitative data on thedistribution of ¹³¹I-TF10.

Imaging Studies Using Pretargeting With TF2 and TF10 BispecificAntibodies and Labeled Peptides

The following studies demonstrate the feasibility of in vivo imagingusing the pretargeting technique with bispecific antibodies and labeledpeptides. While the images were not obtained using an ¹⁸F-metal labeledpeptide as described above, the pretargeting technique with bispecificantibodies may be generally adapted to use with any type of label. Thus,the studies are representative of results that would be obtained usingthe claimed F-18 labeled peptides.

FIG. 20 and FIG. 21 show examples of how clearly delineated tumors canbe detected in animal models using a bsMAb pretargeting method, with an¹¹¹In-labeled di-HSG peptide, IMP-288. In FIG. 20, nude mice bearing 0.2to 0.3 g human pancreatic cancer xenografts were imaged, usingpretargeting with TF10 and ¹¹¹In-IMP-288 peptide. The six animals in thetop of the Figure received 2 different doses of TF10 (10:1 and 20:1 moleratio to the moles of peptide given), and the next day they were givenan ¹¹¹In-labeled diHSG peptide (IMP 288). The 3 other animals receivedonly the ¹¹¹In-IMP-288 (no pretargeting). The images were taken 3 hafter the injection of the labeled peptide and show clear localizationof 0.2-0.3 g tumors in the pretargeted animals, with no localization inthe animals given the ¹¹¹In-peptide alone.

In this study, tumor uptake averaged 20-25% ID/g with tumor/blood ratiosexceeding 2000: 1, tumor/liver ratios of 170: 1, and tumor/kidney ratiosof 18/1. Since tumor uptake shown in the Examples above for theAl-¹⁸F-labeled IMP 449 averaged only about 4-5% ID/g in the same CaPan1xenograft model, it is believed the lower uptake with ¹⁸F-labeledpeptide merely reflects the lower specific activity of theAl-¹⁸F-labeled IMP 449. Nevertheless, the Al-¹⁸F-IMP 449 data show anextraordinary potential for the pretargeted, fluorinated peptide thatwhen combined with the specificity of the TF10 bsMAb would be anexcellent tool for imaging pancreatic or other cancers. Thebiodistribution data for ¹⁸F-labeled peptides far exceed the targetingability of directly radiolabeled antibodies and small engineeredantibody constructs directly labeled with ¹⁸F (Cai et al., J. Nucl. Med.48: 304-310, 2007).

The data shown in FIG. 21 further highlights the sensitivity of thepretargeting method for detecting cancer. Here, a panel of microPETimages was obtained from nude mice injected intravenously with a humancolon cancer cell line and bearing 0.2-0.3 mm micrometastatic tumors inthe lungs. Animals were administered the anti-CEA bsMAb TF2, followedwith a pretargeted ¹²⁴I-labeled peptide. The images show intense uptakein both the transverse and coronal sections at 1.5 h that persisted evenat 21 h. The coronal section is a more posterior view to illustrate thatthe ¹²⁴I-peptide was also seen in the stomach and kidneys 1.5 h afterits injection. The images show what appear to be individual lesions inthe lungs (arrows) that when necropsied were no larger than 0.3 mm indiameter (top panel, transverse sections through the chest) (Sharkey etal., Radiology, 246(2): 497-507, 2008). A control animal pretargetedwith an anti-CD22 TF6 bsMAb and given the same ¹²⁴I-labeled peptide(left side, middle panel) is shown to illustrate the specificity of thelocalization by, in this case, an anti-CEA bsMAb. The coronal section ofthe anti-CEA-pretargeted animals shows the uptake in the chest, as wellas in the kidneys and some activity in the stomach. Significantly, thesame sized lesions in the lungs were not seen in animals given ¹⁸F-FDG.Thus, use of pretargeting antibodies provides greater specificity andsensitivity of detection comparing to the standard F-18-labledfluorodeoxyglucose probe currently used for PET imaging of cancer.

These data further demonstrate the feasibility of imaging usingpretargeting with bispecific antibodies and ¹⁸F-labeled peptides.

Example 13 Synthesis of Folic Acid NOTA Conjugate

Folic acid is activated as described (Wang et. al. Bioconjugate Chem.1996, 7, 56-62.) and conjugated to Boc-NH—CH₂—CH₂—NH₂. The conjugate ispurified by chromatography. The Boc group is then removed by treatmentwith TFA. The amino folate derivative is then mixed with p-SCN-Bn-NOTA(Macrocyclics) in a carbonate buffer. The product is then purified byHPLC. The folate-NOTA derivative is labeled with Al-¹⁸F as described inExample 10 and then HPLC purified. The ¹⁸F-labeled folate is injectedi.v. into a subject and successfully used to image the distribution offolate receptors, for example in cancer or inflammatory diseases (see,e.g., Ke et al., Advanced Drug Delivery Reviews, 56:1143-60, 2004).

Example 14 Pretargeted PET Imaging in Humans

A patient (1.7 m² body surface area) with a suspected recurrent tumor isinjected with 17 mg of bispecific monoclonal antibody (bsMab). The bsMabis allowed to localize to the target and clear from the blood. The F-18labeled peptide (5-10 mCi on 5.7×10⁻⁹ mol) is injected when 99% of thebsMab has cleared from the blood. PET imaging shows the presence ofmicrometastatic tumors.

Example 15 Imaging of Angiogenesis Receptors by F-18 Labeling

Labeled Arg-Gly-Asp (RGD) peptides have been used for imaging ofangiogenesis, for example in ischemic tissues, where α_(v)β₃ integrin isinvolved. (Jeong et al., J. Nucl. Med. 2008, Apr. 15 epub). RGD isconjugated to SCN-Bz-NOTA according to Jeong et al. (2008). Al-¹⁸F isattached to the NOTA-derivatized RGD peptide as described in Example 10above, by mixing aluminum stock solution with F-18 and the derivatizedRGD peptide and heating at 110° C. for 15 min, using an excess ofpeptide to drive the labeling reaction towards completion as disclosedin Example 10. The F-18 labeled RGD peptide is used for in vivobiodistribution and PET imaging as disclosed in Jeong et al. (2008). TheAl-¹⁸F conjugate of RGD-NOTA is taken up into ischemic tissues andprovides PET imaging of angiogenesis.

Example 16 Imaging of Tumors Using F-18 Labeled Bombesin

A NOTA-conjugated bombesin derivative (NOTA-8-Aoc-BBN(7-14)NH₂) isprepared according to Prasanphanich et al. (Proc. Natl. Acad. Sci. USA2007, 104:12462-467). The NOTA-bombesin derivative is labeled withAl-¹⁸F according to Example 10 above. The F-18 labeled bombesinderivative is separated from unlabeled bombesin on an OASIS® column(Waters, Milford, Mass.), as described in Example 10. The Al-¹⁸F labeledNOTA-bombesin conjugate is successfully used for PET imaging ofgastrin-releasing peptide receptor expressing tumors, according toPrasanphanich et al. (2007).

Example 17 Imaging of Tumors Using F-18 Labeled Targetable Conjugates

NOTA derivatives of peptides, polypeptides, proteins, carbohydrates,cytokines, hormones or cell receptor-binding agents are preparedaccording to U.S. Pat. No. 7,011,816 (incorporated herein by referencein its entirety). The NOTA-derivatized targetable conjugates are labeledwith Al-¹⁸F as disclosed in Example 10. The conjugates are administeredin vivo and successfully used for F-18 PET imaging of tumors.

Example 18 Imaging of Tumors Using Bispecific Antibodies

Bispecific antibodies having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable conjugate are prepared according to U.S. Pat. No. 7,052,872,incorporated herein by reference in its entirety. The targetableconjugate comprises one or more NOTA chelating moieties. The targetableconjugate is labeled with Al-¹⁸F as described in Example 10. A subjectwith a disease condition is injected with bispecific antibody. Afterallowing a sufficient time for free bispecific antibody to clear fromthe circulation, the subject is injected with F-18 labeled targetableconjugate. Imaging of the distribution of the F-18 label is performed byPET scanning.

In another exemplary embodiment, humanized or chimeric internalizinganti-CD74 antibody is prepared as described in U.S. Pat. No. 7,312,318.The p-SCN-bn-NOTA precursor is labeled with Al-¹⁸F as described inExample 7. The Al-¹⁸F NOTA is then conjugated to the antibody usingstandard techniques. Upon i.v. injection into a subject with aCD74-expressing tumor, the anti-CD74 antibody localizes to the tumor,allowing imaging of the tumor by PET scanning. In alternativeembodiments, F-18 labeled antibodies are prepared using thealpha-fetoprotein binding antibody Immu31, hPAM4, cPAM4, RS7, anti-CD20,anti-CD19, anti-CEA and anti-CD22, as described in U.S. Pat. Nos.7,300,655; 7,282,567; 7,238,786; 7,238,785; 7,151,164; 7,109,304;6,676,924; 6,306,393 and 6,183,744. The antibodies are conjugated toNOTA using standard techniques and labeled with Al-¹⁸F as described foranti-CD74 antibody. The F-18 labeled antibodies are injected intosubjects and provide successful imaging of tumors by PET scanning.

Example 19 Use of ¹⁸F-Labeled NOTA for Renal Flow Imaging

Aluminum stock solution (20 μL 0.05 M in pH 4 NaOAc buffer) is mixedwith 200 μL of QMA purified F-18 (as in Example 10). The AIF-18 solutionis then mixed with 500 μL pH 4, 0.2 M NOTA and heated for 15 min. Thesample is then diluted in 5 mL PBS for injection. The F-18 labeled NOTAis used directly for successful renal flow imaging.

Example 20 Further Peptide Labeling Studies with Al-¹⁸F

IMP 460 NODA-GA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ was synthesized ina similar manner as described above for IMP 361. The NODA-Ga ligand waspurchased from Chematech and attached on the peptide synthesizer likethe other amino acids. The crude peptide was purified to afford thedesired peptide MH+ 1366.

Radiolabeling of IMP 460

IMP 460 (0.0020 g) was dissolved in 732 μL, pH 4, 0.1 M NaOAc. The F-18was purified as described in Example 10, neutralized with glacial aceticacid and mixed with the Al solution. The peptide solution, 20 μL wasthen added and the solution was heated at 99° C. for 25 min. The crudeproduct was then purified on a Waters HLB column as described above. TheAl-F-18 labeled peptide was in the 1:1 EtOH/H₂O column eluent. Thereverse phase HPLC trace in 0.1% TFA buffers showed a clean single HPLCpeak at the expected location for the labeled peptide.

Example 21 Carbohydrate Labeling

A NOTA thiosemicarbazide derivative is prepared by reacting thep-SCN-bn-NOTA with hydrazine and then purifying the ligand by HPLC.Al-F-18 is prepared as described in Example 10 and the Al-F-18 is addedto the NOTA thiosemicarbazide and heated for 15 min. Optionally theAl-F-18-thiosemicarbazide complex is purified by HPLC. TheAl-F-18-thiosemicarbazide is conjugated to oxidized carbohydrates byknown methods. The F-18 labeled carbohydrate is successfully used forimaging studies using PET scanning.

Example 22 Lipid Labeling

A lipid comprising an aldehyde is conjugated to the Al-F-18 NOTAthiosemicarbazide of Example 21 and the F-18 labeled lipid is used forsuccessful imaging studies using PET scanning.

In an alternative embodiment, a lipid comprising an amino group isreacted with p-SCN-bn-NOTA. The NOTA-labeled lipid is reacted withAl-F-18 as described in the Examples above. The F-18 labeled lipid isused for successful imaging studies using PET scanning.

Example 23 Aptamer Labeling

An aptamer comprising an aldehyde is conjugated to the Al-F-18 NOTAthiosemicarbazide of Example 21. The F-18 labeled aptamer isadministered to a subject and used for successful imaging studies usingPET scanning.

1. An F-18 labeled protein or peptide comprising an F-18 metal complexattached to the protein or peptide.
 2. The F-18 labeled protein orpeptide of claim 1, wherein the F-18 metal complex is bound to achelating moiety attached to the protein or peptide.
 3. The F-18 labeledprotein or peptide of claim 2, wherein the chelating moiety is selectedfrom the group consisting of DOTA, NOTA, DTPA, 2-benzyl-DTPA, TETA,NETA, C-NETA, a macrocyclic polyether, a porphyrin, Tscg-Cys andTsca-Cys.
 4. The F-18 labeled protein or peptide of claim 1, wherein themetal is selected from the group consisting of aluminum, gallium,indium, lutetium and thallium.
 5. The F-18 labeled protein or peptide ofclaim 1, wherein the metal is aluminum.
 6. The F-18 labeled protein orpeptide of claim 1, wherein the F-18 labeled protein or peptide isstable in serum.
 7. The F-18 labeled protein or peptide of claim 2,wherein the peptide is selected from the group consisting of IMP 272,IMP 332, IMP 333, IMP 361, IMP 366, IMP 371, IMP 372, IMP 373, IMP 374,IMP 375, IMP 376, IMP 379, IMP 382, IMP 383, IMP 384, IMP 385, IMP 386,IMP 387, IMP 389 and IMP
 449. 8. The F-18 labeled protein or peptide ofclaim 7, wherein the peptide is IMP 449 (NOTA-ITCbenzyl-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂).
 9. The F-18 labeledprotein or peptide of claim 1, wherein the protein or peptide binds to acell surface receptor.
 10. The F-18 labeled protein or peptide of claim9, wherein the receptor is an angiogenesis receptor and the F-18 labeledprotein or peptide is Al-¹⁸F conjugated RGD-NOTA.
 11. The F-18 labeledprotein or peptide of claim 9, wherein the protein or peptide isbombesin or octreotide.
 12. The F-18 labeled protein or peptide of claim2, wherein the protein or peptide is an antibody or antigen-bindingantibody fragment.
 13. The F-18 labeled protein or peptide of claim 12,wherein the antibody or antigen-binding antibody fragment binds to atumor-associated antigen (TAA).
 14. The F-18 labeled protein or peptideof claim 13, wherein the TAA is selected from the group consisting ofcolon-specific antigen-p (CSAp), carcinoembryonic antigen (CEA), CD4,CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45,CD66a-d, CD67, CD74, CD79a, CD80, CD138, HLA-DR, Ia, Ii, MUC 1, MUC 2,MUC 3, MUC 4, NCA, EGFR, HER 2/neu receptor, TAG-72, EGP-1, EGP-2, A3,KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGFR, EGFR,PDGFR, FGFR, PlGF, ILGF-1, necrosis antigens, IL-2, IL-6, T101 and MAGE.15. The F-18 labeled protein or peptide of claim 13, wherein theantibody or antigen-binding antibody fragment is selected from the groupconsisting of hLL1, hLL2, hlmmu3 1, hPAM4, hRS7, hA19, hA20, hMN-14,hMu-9, hMN-3, hMN-15 and hL243.
 16. A kit for F-18 labeling comprising:a) a metal to form a complex with F-18; and b) a targeting peptidecomprising one or more chelating moieties to bind to the F-18 complex.17. The kit of claim 16, further comprising one or more buffers.
 18. Thekit of claim 16, further comprising a radiolysis protection agent. 19.The kit of claim 18, wherein the radiolysis protection agent is ascorbicacid.
 20. The kit of claim 16, wherein the chelating moiety is selectedfrom the group consisting of DOTA, NOTA, DTPA, 2-benzyl-DTPA, TETA,NETA, C-NETA, a macrocyclic polyether, a porphyrin, Tscg-Cys andTsca-Cys.
 21. The kit of claim 16, wherein the metal is selected fromthe group consisting of aluminum, gallium, indium, lutetium andthallium.
 22. The kit of claim 16, wherein the metal is aluminum. 23.The kit of claim 16, wherein the peptide is selected from the groupconsisting of IMP 272, IMP 332, IMP 333, IMP 361, IMP 366, IMP 371, IMP372, IMP 373, IMP 374, IMP 375, IMP 376, IMP 379, IMP 382, IMP 383, IMP384, IMP 385, IMP 386, IMP 387, IMP 389 and IMP
 449. 24. The kit ofclaim 23, wherein the peptide is IMP
 449. 25. The kit of claim 16,wherein the peptide binds to a cell surface receptor.
 26. The kit ofclaim 25, wherein the receptor is an angiogenesis receptor and the F-18labeled protein or peptide is Al-¹⁸F conjugated RGD-NOTA.
 27. The kit ofclaim 25, wherein the protein or peptide is bombesin or octreotide. 28.The kit of claim 16, further comprising a bispecific antibody orantigen-binding antibody fragment, said antibody with one bindingspecificity for the targeting peptide and another binding specificityfor a target antigen.
 29. The kit of claim 28, wherein the targetantigen is a tumor-associated antigen.
 30. The kit of claim 29, whereinthe target antigen is selected from the group consisting ofcolon-specific antigen-p (CSAp), carcinoembryonic antigen (CEA), CD4,CD5, CD8, CD 14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45,CD66a-d, CD67, CD74, CD79a, CD80, CD138, HLA-DR, Ia, Ii, MUC 1, MUC 2,MUC 3, MUC 4, NCA, EGFR, HER 2/neu receptor, TAG-72, EGP-1, EGP-2, A3,KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGFR, EGFR,PDGFR, FGFR, PlGF, ILGF-1, necrosis antigens, IL-2, IL-6, T101 and MAGE.31. The kit of claim 29, wherein the bispecific antibody orantigen-binding antibody fragment comprises an antibody or fragmentthereof selected from the group consisting of hLL1, hLL2, hlmmu31,hPAM4, hRS7, hA19, hA20, hMN-14, hMu-9, hMN-3, hMN-15 and hL243.
 32. Thekit of claim 28, further comprising a clearing agent to clear bispecificantibody that is not bound to the target antigen from the circulation.33. The kit of claim 29, wherein the bispecific antibody is selectedfrom the group consisting of TF2, TF4, TF10, Mu-9 x 679, MN-14 x 679,LL1 x 679, LL2 x 679, RFB4 x 679, hA20 x 679, RS7 x 679, PAM4 x 679, KC4x 679, MN-3 x 679, MN-15 x 679, Immu31 x 679 and L243 x 679.